Interaction between maternal effect and zygotic effect mutations during maize seed development

Double fertilization of the embryo sac by the two sperm cells of a pollen grain initiates seed development. Proper development of the seed depends not only on the action of genes from the resulting embryo and endosperm, but also on maternal genes acting at two stages. Mutations with both sporophytic maternal effects and gametophytic maternal effects have been identified. A new maternal effect mutation in maize, maternal effect lethal1 (mel1), causes the production of defective seed from mutant female gametophytes. It shows reduced pollen transmission, suggesting a requirement in the male gametophyte, but has no paternal effect on seed development. Interestingly, the defective kernel phenotype of mel1 is conditioned only in seeds that inherit mel1 maternally and are homozygous for the recessive allele (endogenous to the W22 inbred line) of either of two genes, sporophyte enhancer of mel1 (snm1) or snm2, suggesting redundancy between maternally and zygotically required genes. Both mel1 and snm1 map to the short arm of chromosome 2, and snm2 maps to the long arm of chromosome 10. The mode of action of mel1 and the relationship between mel1 and snm1 and snm2 are discussed.     

Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA (MEA) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals.     

DEMETER,a DNA glycosylase domain protein,is required for endosperm gene imprinting and seed viability in arabidopsis

We isolated mutations in Arabidopsis to understand how the female gametophyte controls embryo and endosperm development. For the DEMETER (DME) gene, seed viability depends only on the maternal allele. DME encodes a large protein with DNA glycosylase and nuclear localization domains. DME is expressed primarily in the central cell of the female gametophyte, the progenitor of the endosperm. DME is required for maternal allele expression of the imprinted MEDEA (MEA) Polycomb gene in the central cell and endosperm. Ectopic DME expression in endosperm activates expression of the normally silenced paternal MEA allele. In leaf, ectopic DME expression induces MEA and nicks the MEA promoter. Thus, a DNA glycosylase activates maternal expression of an imprinted gene in the central cell.     

Polycomb group gene function in sexual and asexual seed development in angiosperms

In sexually reproducing angiosperms, double fertilization initiates seed development, giving rise to two fertilization products, the embryo and the endosperm. In the endosperm, a terminal nutritive tissue that supports embryo growth, certain genes are expressed differentially depending on their parental origin, and this genomic imbalance is required for proper seed formation. This parent-of-origin effect on gene expression, called genomic imprinting, is controlled epigenetically through histone modifications and DNA methylation. In the sexual model plant Arabidopsis, the Polycomb group (PcG) genes of the plant Fertilization Independent Seed (FIS)-class control genomic imprinting by specifically silencing maternal or paternal target alleles through histone modifications. Mutations in FIS genes can lead to a bypass in the requirement of fertilization for the initiation of endosperm development and seed abortion. In this review, we discuss the role of the FIS complex in establishing and maintaining genomic imprinting, focusing on recent advances in elucidating the expression and function of FIS-related genes in maize, rice, and Hieracium, and particularly including apomictic Hieracium species that do not require paternal contribution and thus form seeds asexually. Surprisingly, not all FIS-mediated functions described in Arabidopsis are conserved. However, the function of some PcG components are required for viable seed formation in seeds formed via sexual and asexual processes (apomixis) in Hieracium, suggesting a conservation of the seed viability function in some eudicots.     

Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis

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.     

Genetic analysis as a tool to investigate the molecular mechanisms underlying seed development in maize

BACKGROUND: In angiosperms the seed is the outcome of double fertilization, a process leading to the formation of the embryo and the endosperm. The development of the two seed compartments goes through three main phases: polarization, differentiation of the main tissues and organs and maturation. SCOPE: This review focuses on the maize kernel as a model system for developmental and genetic studies of seed development in angiosperms. An overview of what is known about the genetic and molecular aspects underlying embryo and endosperm formation and maturation is presented. The role played by embryonic meristems in laying down the plant architecture is discussed. The acquisition of the different endosperm domains are presented together with the use of molecular markers available for the detection of these domains. Finally the role of programmed cell death in embryo and endosperm development is considered. CONCLUSIONS: The sequence of events occurring in the developing maize seed appears to be strictly regulated. Proper seed development requires the co-ordinated expression of embryo and endosperm genes and relies on the interaction between the two seed components and between the seed and the maternal tissues. Mutant analysis is instrumental in unravelling the genetic control underlying the formation of each compartment as well as the molecular signals interplaying between the two compartments.     

EMBRYO GROWTH AND SEED SIZE IN RAPHANUS SATIVUS: MATERNAL AND PATERNAL EFFECTS IN VIVO AND IN VITRO

Theories on the evolution of the angiosperm seed disagree as to the effects of different plant tissues on embryo growth. To examine the relative contributions of maternal and paternal genes on embryo growth, we conducted controlled crosses in the greenhouse with wild radish plants (Raphanus sativus), looked for maternal, paternal, and interaction effects on embryo development, and compared the performance of embryos within fruits and in embryo culture. Maternal plant identity affected fruit set, seeds per fruit, embryo developmental stage, and mean seed weight. In embryo culture, maternal effects were found for cotyledon size and embryo weight. Paternal effects were fewer or smaller in magnitude than maternal effects. The identity of the pollen donor affected embryo developmental stage and mean seed weight. In culture, paternal effects were detected for cotyledon size and embryo weight. Our results demonstrate that both maternal and paternal elements affect embryo growth. The fact that maternal effects are greater than paternal effects on embryo development in culture may result from cytoplasmic elements or maternal nuclear genes. Embryo performance in vivo compared to that in vitro varied among maternal plants. The interaction between an embryo and its endosperm and maternal tissues may be either positive or negative, depending upon the maternal plant and the embryo's developmental stage.     

A mutational approach to the study of seed development in maize

The maize seed comprises two major compartments, the embryo and the endosperm, both originating from the double fertilization event. The embryogenetic process allows the formation of a well-differentiated embryonic axis, surrounded by a single massive cotyledon, the scutellum. The mature endosperm constitutes the bulk of the seed and comprises specific regions containing reserve proteins, complex carbohydrates, and oils. To gain more insight into molecular events that underlie seed development, three monogenic mutants were characterized, referred to as emp (empty pericarp) on the basis of their extreme endosperm reduction, first recognizable at about 12 d after pollination. Their histological analysis reveals a partial development of the endosperm domains as well as loss of adhesion between pedicel tissues and the basal transfer layer. In the endosperm, programmed cell death (PCD) is delayed. The embryo appears retarded in its growth, but not impaired in its morphogenesis. The mutants can be rescued by culturing immature embryos, even though the seedlings appear retarded in their growth. The analysis of seeds with discordant embryo-endosperm phenotype (mutant embryo, normal endosperm and vice-versa), obtained using B-A translocations, suggests that emp expression in the embryo is necessary, but not sufficient, for proper seed development. In all three mutants the picture emerging is one of a general delay in processes related to growth, as a result of a mutation affecting endosperm development as a primary event.     

Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.     

Arabidopsis haiku mutants reveal new controls of seed size by endosperm

In flowering plants, maternal seed integument encloses the embryo and the endosperm, which are both derived from double fertilization. Although the development of these three components must be coordinated, we have limited knowledge of mechanisms involved in such coordination. The endosperm may play a central role in these mechanisms as epigenetic modifications of endosperm development, via imbalance of dosage between maternal and paternal genomes, affecting both the embryo and the integument. To identify targets of such epigenetic controls, we designed a genetic screen in Arabidopsis for mutants that phenocopy the effects of dosage imbalance in the endosperm. The two mutants haiku 1 and haiku 2 produce seed of reduced size that resemble seed with maternal excess in the maternal/paternal dosage. Homozygous haiku seed develop into plants indistinguishable from wild type. Each mutation is sporophytic recessive, and double-mutant analysis suggests that both mutations affect the same genetic pathway. The endosperm of haiku mutants shows a premature arrest of increase in size that causes precocious cellularization of the syncytial endosperm. Reduction of seed size in haiku results from coordinated reduction of endosperm size, embryo proliferation, and cell elongation of the maternally derived integument. We present further evidence for a control of integument development mediated by endosperm-derived signals.     

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.     

Maternal control of seed development

Maternal control of higher plant seed development is likely to involve female sporophytic as well as female gametophytic genes. While numerous female sporophytic mutants control the production of the ovule and the embryo sac true maternal effect mutations affecting embryo and endosperm development are rare in plants. A new class of female gametophytic mutants has been isolated that controls autonomous development of endosperm. Molecular analyses of these genes, known as FIS class genes, suggest that they repress downstream seed development genes by chromatin remodelling. Expression of the FIS genes in turn is modulated by parent specific expression or genomic imprinting which in turn is controlled by DNA methylation. Thus maternal control of seed development is a complex developmental event influenced by both genetic and epigenetic processes.     

EMBRYONIC FACTOR 31 encodes a tyrosyl-tRNA synthetase that is essential for seed development

Aminoacyl-tRNA synthetases (AARSs) involve the process of catalyzing the ligation of specific amino acids to their cognate tRNAs. Here we identified an Arabidopsis mutant embryonic factor 31 (fac31), its embryos arrested at development from one cell to globular stage. The FAC31 gene was identified by positional cloning and confirmed by a genetic complementation test with two independent T-DNA insertion lines and transgenic rescue with full-length genomic DNA. FAC31 encodes a Tyrosyl-tRNA synthetase and localize to mitochondria and cytoplasm. Fac31 mutants contain a point mutation from CAA to a stop codon TAA which may lead to a truncated protein. The phenotype of fac31 mutants are very similar to the T-DNA insertion lines Salk_016722 and Salk_045570 displayed smaller embryo sac contains only less number of endosperm nucleolus. Genetic analysis showed that the FAC31 gene had no parental effects through the transmission of mutated FAC31 gene by gametes. FAC31 is a high-conserved protein among animals and plants. RT-PCR analysis and promoter-GUS expression showed that it is expressed in nearly all tissues tested, strongly expressed in meristem of seedlings, the primordium of lateral root, young inflorescences, mature pollen, germinated pollen tubes and embryo sacs before heart stage. Our findings suggest that FAC31 is essential for the seed development through regulation the expanding of embryo sac and proliferation of endosperm nucleolus.     

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.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

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

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.