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

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

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.     

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.     

Fitness differences among diploids, tetraploids, and their triploid progeny in Chamerion angustifolium: mechanisms of inviability and implications for polyploid evolution

Abstract. Theoretical models indicate that the evolution of tetraploids in diploid populations will depend on both the relative fitness of the tetraploid and that of the diploid-tetraploid hybrids. Hybrids are believed to have lower fitness due to imbalances in either the ploidy (endosperm imbalance) or the ratio of maternal to paternal genomes in their endosperm (genomic imprinting). In this study we created diploids, tetraploids, and hybrid triploids of Chamerion angustifolium from crosses between field-collected diploid and tetraploid plants and evaluated them at six life stages in a greenhouse comparison. Diploid offspring (from 2 x × 2 x crosses) had significantly higher seed production and lower biomass than tetraploid offspring (from 4 x × 4 x crosses). Relative to the diploid, the cumulative fitness of tetraploids was 0.67. In general, triploids (from 2 x × 4 x , 4 x × 2 x crosses) had significantly lower seed production, lower pollen viability, and higher biomass than diploid individuals. Triploid offspring derived from diploid maternal parents had lower germination rates, but higher pollen production than those with tetraploid mothers. Relative to diploids, the cumulative fitness of 2 x × 4 x triploids and 4 x × 2 x triploids was 0.12 and 0.06, respectively, providing some support for effect of differing maternal:paternal ratios and endosperm development as a mechanism of hybrid inviability. Collectively, the data show that tetraploids exhibit an inherent fitness disadvantage, although the partial viability and fertility of triploids may help to reduce the barrier to tetraploid establishment in sympatric populations.     

Maternal control of nutrient allocation in plant seeds by genomic imprinting

Imprinted genes are commonly expressed in mammalian placentas and in plant seed endosperms, where they exhibit preferential uniparental allelic expression. In mammals, imprinted genes directly regulate placental function and nutrient distribution from mother to fetus; however, none of the >60 imprinted genes thus far reported in plants have been demonstrated to play an equivalent role in regulating the flow of resources to the embryo. Here we show that imprinted Maternally expressed gene1 (Meg1) in maize is both necessary and sufficient for the establishment and differentiation of the endosperm nutrient transfer cells located at the mother:seed interface. Consistent with these findings, Meg1 also regulates maternal nutrient uptake, sucrose partitioning, and seed biomass yield. In addition, we generated an imprinted and nonimprinted synthetic Meg1 ((syn)Meg1) dosage series whereby increased dosage and absence of imprinting both resulted in an unequal investment of maternal resources into the endosperm. These findings highlight dosage regulation by genomic imprinting as being critical for maintaining a balanced distribution of maternal nutrients to filial tissues in plants, as in mammals. However, unlike in mammals, Meg1 is a maternally expressed imprinted gene that surprisingly acts to promote rather than restrict nutrient allocation to the offspring.     

Maternal effects influencing DNA endoreduplication in developing endosperm of Zea mays.

A large proportion of the nuclei in developing endosperm of Zea mays L. undergoes endoreduplication. Nuclear preparations of the entire endosperm from maize kernels of inbred lines, their reciprocal hybrids, and in some cases, F2 and F3 endosperm tissue were evaluated using flow cytometry. Data relative to DNA endoreduplication patterns, percentage of nuclei undergoing endoreduplication, and mean DNA content per nucleus were obtained. The patterns of endoreduplication and extent of DNA amplification differ among some inbreds. In all experiments, the endoreduplication patterns show that the F1 endosperm is more similar to the maternal parent than to the paternal parent. F2 endosperms reveal little difference in endoreduplication patterns among individuals within an F2 family and no more variation than the F1 endosperms. In contrast, F3 endosperms showed greater variation among their endoreduplication patterns. These results indicate a maternal effect on endoreduplication; that is, the genotype of the maternal parent's nuclear genome exerts control over the endoreduplication activities of endosperm tissue.     

Ploidy barrier to endosperm development in maize

Maize kernels inheriting the indeterminate gametophyte mutant (ig) on the female side had endosperms that ranged in ploidy level from diploid (2x) to nonaploid (9x). In crosses with diploid males, only kernels of the triploid endosperm class developed normally. Kernels of the tetraploid endosperm class were half-sized but with well-developed embryos that regularly germinated. Kernels of endosperm composition other than triploid or tetraploid were abortive.-Endosperm ploidy level resulting from mating ig/ig x tetraploid Ig similarly was variable. Most endosperms started to degenerate soon after pollination and remained in an arrested state. Hexaploid endosperm was exceptional; it developed normally during the sequence of stages studied and accounted for plump kernels on mature ears. Since such kernels have diploid maternal tissues (pericarp) but triploid embryos, the present finding favors the view that endosperm failure or success in such circumstances is governed by conditions within the endosperm itself.-Whereas tetraploid endosperm consisting of three maternal genomes and one paternal genome is slightly reduced in size but supports viable seed development, that endosperm having two maternal and two paternal chromosome sets was highly defective and conditioned abortion. Thus, development of maize endosperm evidently is affected by the parental source of its sets of chromosomes.     

Evidence on the nature and origins of endosperm dosage requirements in Solanum and other angiosperm genera

Success of seed development following sexual crosses is primarily dependent on proper endosperm function and development. The failure to produce triploids, or triploid block in 4x×2x crosses served as the impetus for numerous studies of embryo and endosperm to attempt to explain cross failure. Early explanations were based upon a concept of a 232 ploidy balance between maternal tissue, endosperm, and embryo. Subsequent studies done with maize demonstrated that normal endosperm development in intraspecific maize crosses is dependent solely on having a 21 maternal to paternal genome dosage in the endosperm. These results have been modified and extended to solanaceous species in the form of an endosperm dosage system in which empirically determined factors must bear the same 21 relationship for crosses to succeed. Crossing behavior of these species suggest that the system is polygenically controlled and regulates both interspecific and intraspecific crosses. Endosperm dosage systems explain many aspects of species evolution, but the system appears to have originated as an ancient means of ensuring diploid fidelity.     

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.     

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.     

Extensive maternal DNA hypomethylation in the endosperm of Zea mays

A PCR-based genomic scan has been undertaken to estimate the extent and ratio of maternally versus paternally methylated DNA regions in endosperm, embryo, and leaf of Zea mays (maize). Analysis of several inbred lines and their reciprocal crosses identified a large number of conserved, differentially methylated DNA regions (DMRs) that were specific to the endosperm. DMRs were hypomethylated at specific methylation-sensitive restriction sites upon maternal transmission, whereas upon paternal transmission, the methylation levels were similar to those observed in embryo and leaf. Maternal hypomethylation was extensive and offers a likely explanation for the 13% reduction in methyl-cytosine content of the endosperm compared with leaf tissue. DMRs showed identity to expressed genic regions, were observed early after fertilization, and maintained at a later stage of endosperm development. The implications of extensive maternal hypomethylation with respect to endosperm development and epigenetic reprogramming will be discussed.     

Increased Maternal Genome Dosage Bypasses the Requirement of the FIS Polycomb Repressive Complex 2 in Arabidopsis Seed Development

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.     

When genomes collide: aberrant seed development following maize interploidy crosses

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

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

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

Dosage effects in the endosperm of diplosporous apomictic Tripsacum (Poaceae)

 Imprinting in the endosperm of angiosperms, a phenomena by which expression of alleles differs depending on whether they originate from the male or female parent, has been shown to explain most failure of interploidy or interspecific crosses in plants. Because of imprinting, seeds develop normally only if a specific dosage is represented in the endosperm, with the relative contributions of genomes in the ratio of two maternal doses to one paternal dose (2m:1p). In Tripsacum, a wild relative of maize, all polyploids reproduce through the diplosporous type of apomixis. Diplospory results from meiotic failure in megasporocytes that develop into eight-nucleate unreduced female gametophytes. The male gametophytes remain unaffected. Flow cytometry was used to determine ploidy levels in the endosperm of both apomictic and sexual Tripsacum accessions. In both cases, fertilization appeared to involve only one sperm nucleus. Therefore, endosperm of apomictic Tripsacum develops normally even though the ratio of genomic contributions deviates from the normal 2m:1p ratio. Ratios of 2:1, 4:1, 4:2, 8:1 and 8:2 were observed, depending on both the ploidy level of the parents and the mode of reproduction. Thus, specific dosage effects are seemingly not required for endosperm development in Tripsacum. These findings suggest that evolution of diplosporous apomixis might have been restricted to species with few or no imprinting requirements, and the findings have strong implications regarding the transfer of apomixis to sexually reproducing crops. Received: 17 February 1997 / Revision accepted: 7 July 1997