Chromatin-remodeling factors regulate the establishment of transcriptional programs during plant development. Although 42 genes encoding members of the SWI2͞SNF2 family have been identified in Arabidopsis thaliana, <10 have been assigned a precise function on the basis of a mutant phenotype, and none have been shown to play a specific role during the gametophytic phase of the plant life cycle. A. thaliana chromatin-remodeling protein 11 (CHR11) encodes an imitation of switch (ISWI)-like chromatin-remodeling protein abundantly expressed during female gametogenesis and embryogenesis in Arabidopsis. To determine the function of CHR11 in wild-type plants, we introduced a hairpin construct leading to the production of double-stranded RNA, which specifically degraded the endogenous CHR11 mRNA by RNA interference (RNAi). Transcription of the RNAi-inducing hairpin RNA was driven by either a constitutive cauliflower mosaic virus 35S promoter (CaMV35S) acting at most stages of the sporophytic phase or a newly identified specific promoter acting at the onset of the female gametophytic phase (pFM1). All adult transformants that constitutively lacked sporophytic CHR11 activity showed reduced plant height and small cotyledonary embryos with limited cell expansion. In contrast, RNAi lines in which CHR11 was specifically silenced at the onset of female gametogenesis (megagametogenesis) had normal height and embryo size but had defective female gametophytes arrested before the completion of the mitotic haploid nuclear divisions. These results show that CHR11 is essential for haploid nuclear proliferation during megagametogenesis and cell expansion during the sporophytic phase, demonstrating the functional versatility of SWI2͞SNF2 chromatinremodeling factors during both generations of the plant life cycle.imitation of switch proteins ͉ megagametophyte ͉ RNA interference ͉ seed development ͉ functional megaspore T he accessibility of DNA to transcription factors or other types of interacting molecules is regulated by enzymatic complexes that modify nucleosomal structure by means of ATP-dependent chromatin-remodeling or histone modification (1). ATPdependent chromatin-remodeling factors are multisubunit complexes that alter the chromatin structure by changing the conformational state of the nucleosome. These structural changes are accomplished without covalent modification and can be involved in either the activation or the repression of transcription (2). Members of the SWI2͞SNF2 family of ATP-dependent proteins share an ATPase domain that is essential for their chromatin-remodeling activity. In addition, SWI2͞SNF2 proteins have a large variety of Nand C-terminal domains that are often involved in their interaction with other members of specific chromatin-associated complexes. The largest eukaryotic group of SWI2͞SNF2, ATP-dependent, chromatin-remodeling proteins is the imitation of switch (ISWI) subfamily. Originally identified in Drosophila (3, 4), ISWI members are distinguished from other SWI2͞SNF2 proteins by the presence of t...
In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants.
Each year, plants and animals perform the task of repopulating the planet through patterns of courtship and mating that have a unifying and compelling logic: the production of offspring. Although life of nearly all organisms is organized around sex and breeding, Darwinian thinking focused more on the struggle for existence than on evolutionary significance of this frantic race to reproduce. In Darwin's own words, "We do not know the final cause of sexuality; why new beings should be produced by the union of the two sexual elements. The whole subject is hidden in darkness. . ." (Darwin 1862). In plants, a major consequence of this search for survival is the evolution of a multitude of reproductive alternatives that have intrigued botanists, geneticists, and evolutionary biologists for more than 100 years. Because sexually derived genetic diversity is interpreted as essential for adaptation, it is often thought that sex is necessary for the perpetuation of a species; however, many organismsincluding several hundred families of flowering plants-are going efficiently about propagating their kind without bothering with meiosis and mating. Whereas many plants can undergo vegetative propagation, through the production of stolons, bulbs, or rhizomes, for example, many others have developed methods to produce an embryo from a single cell whose nucleus is not formed by the fusion of two gametes, offering a direct developmental and evolutionary challenge to sexual reproduction. Recent evidence suggests that epigenetic mechanisms that control transcriptional silencing of DNA repetitive elements and heterochromatin are crucial for the acquisition of cell identity in the ovule, opening the possibility that the developmental distinction between sexual development and apomixis might have evolved as an adaptive response to evolutionary forces that modulate structural variation and reproductive versatility in flowering plants.The emergence of all plants is derived from a common ancestor that endosymbiotically acquired a photosynthetic bacterium capable of transforming light into energy, an evolutionary event that changed the surface of the planet by progressively transforming an environment dominated by cyanobacteria (blue-green bacteria) into a habitat predominantly occupied by photosynthetic multicellular organisms. Although fossilized spores suggest that land plants appeared more than 450 million years ago, the establishment of their life cycle through the alternation of haploid and diploid generations remains an unsolved mystery. In contrast to green algae and mosses, in which the gametophytic (haploid) phase is free-living and temporally dominant, flowering plants have a reduced and ephemeral gametophyte that forms male and female gametes and is necessary to ensure sexual reproduction.Plants have evolved a characteristic life strategy with alternating diploid and haploid phases (or generations), continuous postembryonic development that derives in the formation of aerial and underground meristems, and the absence of a germli...
IntroductionAlthough DNA methylation patterns are generally considered to be faithfully inherited in Arabidopsis thaliana (Arabidopsis), there is evidence of reprogramming during both male and female gametogenesis. The gynoecium is the floral reproductive organ from which the ovules develop and generate meiotically derived cells that give rise to the female gametophyte. It is not known whether the gynoecium can condition genomic methylation in the ovule or the developing female gametophyte.MethodsWe performed whole genome bisulfite sequencing to characterize the methylation patterns that prevail in the genomic DNA of pre-meiotic gynoecia of wild-type and three mutants defective in genes of the RNA-directed DNA methylation pathway (RdDM): ARGONAUTE4 (AGO4), ARGONAUTE9 (AGO9), and RNA-DEPENDENT RNA POLYMERASE6 (RDR6). ResultsBy globally analyzing transposable elements (TEs) and genes located across the Arabidopsis genome, we show that DNA methylation levels are similar to those of gametophytic cells rather than those of sporophytic organs such as seedlings and rosette leaves. We show that none of the mutations completely abolishes RdDM, suggesting strong redundancy within the methylation pathways. Among all, ago4 mutation has the strongest effect on RdDM, causing more CHH hypomethylation than ago9 and rdr6. We identify 22 genes whose DNA methylation is significantly reduced in ago4, ago9 and rdr6 mutants, revealing potential targets regulated by the RdDM pathway in premeiotic gyneocia. DiscussionOur results indicate that drastic changes in methylation levels in all three contexts occur in female reproductive organs at the sporophytic level, prior to the alternation of generations within the ovule primordium, offering a possibility to start identifying the function of specific genes acting in the establishment of the female gametophytic phase of the Arabidopsis life cycle.
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