In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC)1,2. Because some mutants and certain sexual species exhibit more than one MMC2-4, and many others are able to form gametes without meiosis (by apomixis)5, it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to play an important function in determination6. Here we show that the Arabidopsis protein ARGONAUTE9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING3 and RNA-DEPENDENT RNA POLYMERASE6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNA) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24 nucleotide (nt) sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA–dependent silencing in plant gametes.

Defining the contributions and interactions of paternal and maternal genomes during embryo development is critical to understand the fundamental processes involved in hybrid vigor, hybrid sterility, and reproductive isolation. To determine the parental contributions and their regulation during Arabidopsis embryogenesis, we combined deep-sequencing-based RNA profiling and genetic analyses. At the 2-4 cell stage there is a strong, genome-wide dominance of maternal transcripts, although transcripts are contributed by both parental genomes. At the globular stage the relative paternal contribution is higher, largely due to a gradual activation of the paternal genome. We identified two antagonistic maternal pathways that control these parental contributions. Paternal alleles are initially downregulated by the chromatin siRNA pathway, linked to DNA and histone methylation, whereas transcriptional activation requires maternal activity of the histone chaperone complex CAF1. Our results define maternal epigenetic pathways controlling the parental contributions in plant embryos, which are distinct from those regulating genomic imprinting.

Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference-mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.

contributed equally to this workThe struwwelpeter (swp) mutant in Arabidopsis shows reduced cell numbers in all aerial organs. In certain cases, this defect is partially compensated by an increase in ®nal cell size. Although the mutation does not affect cell cycle duration in the young primordia, it does in¯uence the window of cell proliferation, as cell number is reduced during the very early stages of primordium initiation and a precocious arrest of cell proliferation occurs. In addition, the mutation also perturbs the shoot apical meristem (SAM), which becomes gradually disorganized. SWP encodes a protein with similarities to subunits of the Mediator complex, required for RNA polymerase II recruitment at target promoters in response to speci®c activators. To gain further insight into its function, we overexpressed the gene under the control of a constitutive promoter. This interfered again with the moment of cell cycle arrest in the young leaf. Our results suggest that the levels of SWP, besides their role in pattern formation at the meristem, play an important role in de®ning the duration of cell proliferation. Keywords: leaf development/Mediator complex/ primordia/shoot apical meristem IntroductionIn multicellular animals and plants, body size is often correlated with cell number, and it has been suggested that the strict control of cell division is an important factor in the regulation of growth and development (Raff, 1996;Conlon and Raff, 1999;Vernoux et al., 2000c). However, other views exist, based on cases where substantial alterations in cell proliferation do not alter organ size or developmental patterns. In imaginal wing discs of Drosophila, for example, the overexpression of E2F, a transcription factor promoting cell division, leads to increased cell number, but the ®nal wing size remains normal as the cells are smaller. Conversely, overexpression of the Drosophila homologue of the retinoblastoma protein reduces cell numbers, but again wing size is not affected (reviewed in Day and Lawrence, 2000;Weinkove and Leevers, 2000). Similar observations have been made for plants. Tobacco expressing a dominant-negative version of the Arabidopsis cell cycle regulator CDKA (cdc2aAt) showed almost normally sized leaves with fewer and larger cells (Hemerly et al., 1995). More recently, overexpression of inhibitors of cell division, the so-called KIP-related proteins (KRPs), reduced cell numbers in Arabidopsis leaves, a defect that was largely compensated by increased cell size (Wang et al., 2000;De Veylder et al., 2001). Together, these observations suggest that intrinsic mechanisms exist, operating throughout the organ or organism as a unit and dictating size and shape. According to such a scenario, cell proliferation or expansion itself would not be strictly regulated in time and space by a complex network of instructions given to the individual cells, but would simply follow growth patterns coordinated at the tissue level (Kaplan and Hagemann, 1991; Potter and Xu, 2001). The molecular basis underlying such a...

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host-symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant-fungal and plant-bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.actinorhizal symbioses ͉ Casuarina glauca ͉ mycorrhizae ͉ signaling R oot endosymbioses are associations between plants and soil microorganisms involving intracellular accommodation of microbes within host cells. The most widespread of these associations is arbuscular mycorrhiza (AM), which is formed by the majority of land plants with fungi belonging to the phylum Glomeromycota (1). In contrast, nitrogen-fixing nodulation symbioses of plant roots and bacteria are restricted to four orders of eurosid dicots (2). Actinorhiza, formed by members of the Fagales, Rosales, and Cucurbitales with Gram-positive Frankia bacteria, differs from the interaction of legumes with Gramnegative rhizobia in several morphological and cytological aspects (3). Although these differences suggest independent regulatory mechanisms, the close relatedness of nodulating lineages indicates a common evolutionary basis of root nodulation symbioses (2). In the legume-rhizobia interaction, among the key factors mediating recognition between the plant and the bacteria are Nod factors (NFs). NFs are bacterial lipochitooligosaccharides with an N-acetylglucosamine backbone (4). The perception of NFs induces a series of responses in host roots, including ion flux changes and membrane depolarization, rhythmic calcium oscillations in and around the nucleus (calcium spiking), cytoskeletal modifications and root hair curling, and activation of cortical cell divisions (5). Extensive mutant screenings performed in legumes led to the identification of several loci involved in this signaling cascade, and recently most of the corresponding genes were identified by map-based approaches (6). In Lotus japonicus, two genes, NFR1 and NFR5 encoding receptor-like serine/threonine kinases with L...

Plants and animals have each evolved different reproductive strategies that determine the extent to which the parents, usually the female, exert control over the developing offspring. Fertilization can be external or internal. In the latter case, the fertilized egg can be laid on a nutritive substrate (e.g., insects) or it develops within a protective and nurturing matrix (the placenta of mammals, the egg of birds and reptiles, the seed of seed plants). The MZT marks the end of maternal control and the onset of zygotic control over embryo development. It is characterized by the simultaneous degradation of stored maternal products and zygotic genome activation (Andéol 1994;Schultz 2002;Tadros et al. 2003;Stitzel and Seydoux 2007). In animals, the MZT occurs several cell cycles after fertilization. As a consequence, the first stages of embryonic development essentially rely on maternally deposited products. Despite divergent reproductive strategies and differences in the parental provisioning for offspring development, current evidence indicates that plants and animals share a marked maternal control of early postfertilization development. We review this evidence and our knowledge regarding the mechanisms controlling the onset of the MZT in both animals and plants. We hypothesize that shared biological constraints on early embryo development explain the existence of convergent mechanisms for maintaining maternal predominance. EARLY EMBRYOGENESIS IN ANIMALS AND HIGHER PLANTSEmbryogenesis in flowering plants and animals has in common the union of a male and a female gamete that produces the zygote and the sequential events of cell proliferation, morphogenesis, and organogenesis that follow ( Fig. 1). In animals, a single female gamete, the oocyte, fuses with a motile sperm to produce the zygote. Cell proliferation generates a multicellular morula of 16-32 cells in mammals, a blastula of approximately 30,000 cells in Xenopus, and a syncytial blastoderm of about 6000 nuclei in Drosophila. Cellularization (for the syncytial blastoderm of insects) and asymmetric divisions define the onset of morphogenesis, and cellular migration marks the beginning of gastrulation and organogenesis (Browder et al. 1991).In flowering plants, two pairs of gametes fuse, a process termed double fertilization. The two female gametes, the egg and central cell, are each fertilized by a sperm to produce the embryo and the endosperm, respectively. The endosperm is a protective and nurturing tissue that has a role similar to that of the placenta in eutherian mammals (Harper et al. 1970). The female gametes are produced together with "accessory" cells (the synergids and antipodals) within the female gametophyte (embryo sac), which is enclosed in the ovule that, after fertilization, forms the seed. Ovule and seed development occurs in the gynoecium in the center of the flower. The two sperms are produced by the male gametophyte (pollen), which germinates and grows through tissues of the gyneocium to deliver the sperm cells to the embryo sac. In pla...

Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency and to prevent transgenerational inheritance of epimutations. However, the extent of DNA methylation reprogramming in plants remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DOMAINS REARRANGED METHYLASE 2 (DRM2) and RNA polymerase V (Pol V), two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single-cell level with high temporal resolution and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA, as we illustrate in mouse embryonic stem cells.

SummaryT-DNA integration in the nuclear plant genome may lead to rearrangements of the plant target site. Here we present evidence for a chromosomal inversion of 26 cM bordered by two T-DNAs in direct orientation, which is linked to the mgoun2 mutation. The integration sites of the T-DNAs map at positions 80 and 106 of chromosome I and we show that each T-DNA is bordered by plant sequences from positions 80 and 106, respectively. Although the T-DNAs are physically distant, they are genetically closely linked. In addition, three markers located on the chromosome segment between the two T-DNA integration sites show no recombination with the mgo2 mutation. We show that the inversion cannot be a consequence of a recombination event between the two T-DNAs, but that the integration of the T-DNAs and the inversion were two temporally linked events. T-DNA integration mechanisms that could have led to this inversion are discussed.