Project description:The endosperm is an ephemeral tissue that nourishes the developing embryo, similar to the placenta in mammals. In most angiosperms, endosperm development starts as a syncytium, in which nuclear divisions are not followed by cytokinesis. The timing of endosperm cellularization largely varies between species, and the event triggering this transition remains unknown. Here we show that increased auxin biosynthesis in the endosperm prevents its cellularization, leading to seed arrest. Auxin-overproducing seeds phenocopy paternal-excess triploid seeds derived from hybridizations of diploid maternal plants with tetraploid fathers. Concurrently, auxin-related genes are strongly overexpressed in triploid seeds, correlating with increased auxin activity. Reducing auxin biosynthesis and signaling reestablishes endosperm cellularization in triploid seeds and restores their viability, highlighting a causal role of increased auxin in preventing endosperm cellularization. We propose that auxin determines the time of endosperm cellularization, and thereby uncovered a central role of auxin in establishing hybridization barriers in plants.
Project description:The endosperm is a reproductive tissue supporting embryo development. In most 30 flowering plants, the initial divisions of endosperm nuclei are not succeeded by 31 cellularization; this process occurs only after a specific number of mitotic cycles have 32 taken place. The timing of cellularization significantly influences seed viability and size. 33 Previous research implicated auxin as a key factor in initiating nuclear divisions and 34 determining the timing of cellularization. Here, we uncover the involvement of a family 35 of clustered auxin response factors (cARFs) as dosage-sensitive regulators of 36 endosperm cellularization. cARFs, maternally expressed and paternally silenced, are 37 shown to induce cellularization, thereby restricting seed growth. Our findings align with 38 the predictions of the parental conflict theory, suggesting that cARFs represent major 39 molecular targets in this conflict. We further demonstrate a recurring amplification of 40 cARFs in the Brassicaceae, suggesting an evolutionary response to parental conflict 41 by reinforcing maternal control over endosperm cellularization. Our study highlights 42 that antagonistic parental control on endosperm cellularization converges on auxin 43 biosynthesis and signaling.
Project description:Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of siRNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo. Keywords: Epigenetics; bisulfite sequencing Examination of DNA methylation in four Arabidopsis tissues Description of processed data and raw data file contents can be found in the attached README.txt file
Project description:Laser capture microdissection (LCM) provides a useful method for isolating specific cells or tissues from biological samples. Here, we adapted microdissection protocols to allow high-resolution transcript analysis of different tissues from developing Arabidopsis seed. However, to obtain enough RNA for microarray analyses it was necessary to amplify the RNA. Microarray analyses, using endosperm derived RNA amplified by two-round IVT, reproducibly identified endosperm enriched marker genes. Keywords: LCM Endosperm Arabidopsis
Project description:Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of siRNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo. Keywords: Epigenetics; bisulfite sequencing
Project description:To obtain more information on auxin-regulated gene expression, we treated Arabidopsis seedlings with auxin biosynthesis or signaling inhibitors, then performed DNA microarray analyses.
Project description:We found that auxin stimulates gene expression of DWF4, which encodes a rate-dertermining step in brassinosteroid biosynthesis pathways. This increased gene expressioin subsequently led to elevation of the biosynthetic flux in Arabidopsis roots. To determine the list of genes that are regulated by auxin-synthesizing brassinosteroids, we challenged Arabidopsis seedlings with either auxin only or auxin plus brassinosteroid biosynthetic inhibitor brassinazole. Keywords: Hormone treatment
Project description:Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. Profiling the endosperm epigenetic landscape on a genome-wide scale is challenging due to its small size, mode of development, and close association with maternal tissue. Here, we applied a low input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using low numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN makes it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable the study of tissue or even cell-type specific epigenomes in Arabidopsis and other plant species.