The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis.

The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1), was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring.

Transcriptional profiles underlying parent-of-origin effects in seeds of Arabidopsis thaliana.

BACKGROUND: Crossing plants of the same species but different ploidies can have dramatic effects on seed growth, but little is known about the alterations to transcriptional programmes responsible for this. Parental genomic imbalance particularly affects proliferation of the endosperm, with an increased ratio of paternally to maternally contributed genomes ('paternal excess') associated with overproliferation, while maternal excess inhibits endosperm growth. One interpretation is that interploidy crosses disrupt the balance in the seed of active copies of parentally imprinted genes. This is supported by the observation that mutations in imprinted FIS-class genes of Arabidopsis thaliana share many features of the paternal excess phenotype. Here we investigated gene expression underlying parent-of-origin effects in Arabidopsis through transcriptional profiling of siliques generated by interploidy crosses and FIS-class mutants. RESULTS: We found that fertilized fis1 mutant seeds have similar profiles to seeds with paternal excess, showing that the shared phenotypes are underpinned by similar patterns of gene expression. We identified genes strongly associated with enhanced or inhibited seed growth; this provided many candidates for further investigation including MADS-box transcription factors, cell cycle genes, and genes involved in hormone pathways. CONCLUSIONS: The work presented here is a step towards understanding the effects on seed development of the related phenomena of parental genome balance and imprinting.

Yield assessment of integument-led seed growth following targeted repair of auxin response factor 2.

SUMMARY: It is becoming increasingly vital to improve the yield of seed crops to feed an expanding population and, more recently, for biofuel production. One strategy to increase the yield is to increase the seed size, provided that there is not a concomitant decrease in seed number. In a previous study, we described a mutant in the auxin response factor 2 (ARF2) gene which produced extra cells in the seed coat and, subsequently, enlarged seeds. However, arf2 mutant plants also show severely reduced self-fertility caused, in part, by over-elongated sepals that prevent flower opening. As a low seed set increases individual seed size, a meaningful comparison of the yield in arf2 and wild-type plants could not be conducted. In this study, we show that targeted expression of wild-type ARF2 in the sepals and petals of arf2-9 mutant flowers restores flower opening and dramatically increases seed set. The restored plants retain both enlarged integuments and increased seed size, reinforcing previous evidence that arf2 mutations increase seed weight through their effect on integuments and not only via reduced fertility. We also show that the measurement of the harvest index in Arabidopsis is useful in assessing the impact of introduced traits on the yield.

Epigenetics: imprinting in plants and mammals--the same but different?

Plants and animals both exhibit parental imprinting, but do they control it the same way? Recent studies show that in Arabidopsis, as in mammals, imprinted alleles are subject to DNA methylation--but, surprisingly, the default state is silence rather than activity.

Proliferative phase endosperm promoters from Arabidopsis thaliana.

Endosperm accounts for a large proportion of human nutrition and is also a major determinant of seed viability and size, not only in cereals, but also in species with ephemeral endosperms, such as soybean and oilseed rape. The extent of endosperm proliferation early in seed development is a crucial component in setting seed size; therefore, a biotechnological approach for the modification of this trait requires promoters active in early endosperm. To find such promoters, we constructed an array based on cDNAs extracted from developing Arabidopsis seeds enriched for proliferating endosperm. Hybridization with RNA extracted from vegetative and reproductive tissues, including endosperm, and subsequent data filtering yielded sets of endosperm-expressed and endosperm-preferred genes, including many hundreds not previously identified in array experiments designed to detect genes expressed in Arabidopsis seeds. Of eight promoters selected for validation, seven were active in early endosperm, three with no detected activity elsewhere in the plant. Therefore, this strategy has yielded proliferative phase endosperm promoters which should be useful in altering seed size.

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.

Double fertilization in Arabidopsis thaliana involves a polyspermy block on the egg but not the central cell.

In animal reproduction, thousands of sperm may compete to fertilize a single egg, but polyspermy blocks prevent multiple fertilization that would otherwise lead to death of the embryo. In flowering plants, successful seed development requires that only two sperm are delivered to the embryo sac, where each must fertilize a female gamete (egg or central cell) to produce the embryo and endosperm. Therefore, polyspermy must be avoided, not only to prevent abnormalities in offspring, but to ensure double fertilization. It is not understood how each sperm fertilizes only one female gamete, nor has the existence of polyspermy barriers been directly tested in vivo. Here, we sought evidence for polyspermy blocks in angiosperms using the polyspermic tetraspore (tes) mutant of Arabidopsis, which allows in-vivo challenge of egg and central cell with multiple male gametes. We show that tes mutant pollen tubes can transmit more than one sperm pair to an embryo sac, and that sperm from more than one pair can participate in fertilization. We detected endosperms but not embryos with ploidies that could only result from multiple fertilization. Our results therefore demonstrate an in-vivo polyspermy block on the egg, but not the central cell of a flowering plant.

MATERNALLY EXPRESSED PAB C-TERMINAL, a novel imprinted gene in Arabidopsis, encodes the conserved C-terminal domain of polyadenylate binding proteins.

Parental imprinting is important for seed development, but few imprinted genes have been identified in plants. The four known imprinted genes in Arabidopsis thaliana encode transcriptional regulators. Here, we describe a novel imprinted gene, MATERNALLY EXPRESSED PAB C-TERMINAL (MPC), which encodes the C-terminal domain of poly(A) binding proteins (PABPs). PABPs play roles in mRNA stability and translation. MPC interacts with proteins that also interact with the C-terminal domain of typical PABPs, suggesting that MPC may regulate translation by modulating PABP activity. In the endosperm, MPC is expressed only from the maternal allele. Reduction of MPC expression affects seed development. In dna methyltransferase1 (met1) mutants, MPC is ectopically expressed, and the paternal allele is active in the endosperm. CGs in the 5' flanking region and gene body of MPC lose methylation in a met1 background. Both regions are required to confer imprinted reporter expression, suggesting that the gene body contains imprinting control region elements. In Arabidopsis, DEMETER (DME) activates expression of maternal alleles. MPC expression is reduced in flowers and seeds in a dme-4 mutant but only after fertilization in dme-1. We conclude that other factors along with DME promote MPC expression and that DME has indirect effects on imprinted gene expression in endosperm.

Deeper into the maize: new insights into genomic imprinting in plants.

Current models for regulation of parent-specific gene expression in plants have been based on a small number of imprinted genes in Arabidopsis. These present repression as the default state, with expression requiring targeted activation. In general, repression is associated with maintenance methylation of cytosines, while no role has been found in Arabidopsis imprinting for de novo methylation--unlike the case in mammals. A recent paper both reinforces and challenges the model drawn from Arabidopsis. Methylation patterns of two imprinted loci in maize were tracked from gametes to offspring, enabling an exploration of the timing of imprinting. For one gene, fie1, the results were as expected: parent-specific methylation patterns were inherited from the three types of gamete: egg, central cell and sperm. The behaviour of fie2, however, was a surprise: no alleles were methylated in the gametes, although paternally contributed fie2 is methylated and silent in the endosperm, indicating that, in some cases, plant imprinting requires de novo DNA methylation. This work significantly broadens our understanding of plant imprinting and points to a greater diversity in imprinting mechanisms than has previously been appreciated.

The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs.

Control of seed size involves complex interactions among the zygotic embryo and endosperm, the maternally derived seed coat, and the parent plant. Here we describe a mutant in Arabidopsis, megaintegumenta (mnt), in which seed size and weight are dramatically increased. One factor in this is extra cell division in the integuments surrounding mnt mutant ovules, leading to the formation of enlarged seed coats. Unusually for integument mutants, mnt does not impair female fertility. The mnt lesion also has pleiotropic effects on vegetative and floral development, causing extra cell division and expansion in many organs. mnt was identified as a mutant allele of AUXIN RESPONSE FACTOR 2 (ARF2), a member of a family of transcription factors that mediate gene expression in response to auxin. The mutant phenotype and gene expression studies described here provide evidence that MNT/ARF2 is a repressor of cell division and organ growth. The mutant phenotype also illustrates the importance of growth of the ovule before fertilization in determining final size of the seed.

Genetic mechanisms of apomixis.

The introduction of apomixis to crops would allow desirable genotypes to be propagated while preventing undesirable gene flow, but so far there has been little success in transferring this trait from a natural apomict to another species. One explanation is the sensitivity of endosperm to changes in relative maternal and paternal contribution owing to parental imprinting, an epigenetic system of transcriptional regulation by which some genes are expressed from only the maternally or paternally contributed allele. In sexual species, endosperm typically requires a ratio of two maternal genomes to one paternal genome for normal development, but this ratio is often altered in apomicts, suggesting that the imprinting system is altered as well. We present evidence that modification of DNA methylation is one mechanism by which the imprinting system could be altered to allow endosperm development in apomicts. Another feature of natural apomixis is the modification of the normal fertilization programme. Sexual reproduction uses both sperm from each pollen grain, but pseudogamous apomicts, which require a sexual endosperm to support the asexual embryo, often use just one. We present evidence that multiple fertilization of the central cell is possible in Arabidopsis thaliana, suggesting that pseudogamous apomicts may also need to acquire a mechanism for preventing more than one sperm from contributing to the endosperm. We conclude that strategies to transfer apomixis to crop species should take account of endosperm development and particularly its sensitivity to parental imprinting, as well as the mechanism of fertilization.

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.

Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation.

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.