Transcription profiling of Arabidopsis seed development in LEAFY COTYLEDON1 mutants
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ABSTRACT: Analysis of gene expression in Ws-0 lec1 (LEAFY COTYLEDON1) mutant Arabidopsis thaliana. Developmental stages studied includes 24-Hr post-fertilization, globular stage, cotyledon stage, mature green stage, post-mature green stage, and seedlings. Microarray Methods: Total RNA was extracted using the Hot Borate Method [Stones et. al. PNAS vol 98 no 20: 11806-11811 (2001)]. Biotinylated cRNA were prepared using the ENZO BioArray High Yield RNA Transcript Labeling Kit (Farmingdale, NY). cRNA was subsequently hybridized to Affymetrix ATH1 Arabidopsis GeneChips. The scanned array images were analyzed using Affymetrix Microarray Suite 4.0 (MAS 4.0) with a global scaling intensity set at 500.
Project description:Analysis of gene expression in Ws-0 lec1 (LEAFY COTYLEDON1) mutant Arabidopsis thaliana. Developmental stages studied includes 24-Hr post-fertilization, globular stage, cotyledon stage, mature green stage, post-mature green stage, and seedlings. Microarray Methods: Total RNA was extracted using the Hot Borate Method [Stones et. al. PNAS vol 98 no 20: 11806-11811 (2001)]. Biotinylated cRNA were prepared using the ENZO BioArray High Yield RNA Transcript Labeling Kit (Farmingdale, NY). cRNA was subsequently hybridized to Affymetrix ATH1 Arabidopsis GeneChips. The scanned array images were analyzed using Affymetrix Microarray Suite 4.0 (MAS 4.0) with a global scaling intensity set at 500. Keywords: other
Project description:LEAFY COTYLEDON1 (LEC1), a subunit of the NF-Y CCAAT binding transcription factor, is a key regulator that controls many aspects of seed development including embryo growth and seed storage accumulation. To identified genomic region bound by LEC1 and target genes of LEC1, we expressed a FLAG-tagged form of LEC1 under the control of its native promoter and terminator in lec1-4 mutant seeds. Using an anti-FLAG antibody (Sigma), we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using chromatin isolated from mature stage seeds to identify the genes bound by LEC1. These data reveal that the transcription regulation network of LEC1.
Project description:LEAFY COTYLEDON1 (LEC1), expressed in both endosperm and embryo, acts as a central regulator that controls diverse aspects of seed development including embryogenesis and seed maturation. LEC1 expression in the endosperm can be detected within one day after fertilization. The gene networks and developmental processes controlled by LEC1 in endosperm remain unclear. Here, we applied a modified INTACT system to purify endosperm nuclei and used for ChIP-seq to identify the genome-wide occupancy of LEC1 in the developing endosperms at three stages, namely nuclei proliferation, cellularization, and degeneration. The ChIP-seq in combination with transcriptome analyses indicated a regulatory role of LEC1 in controlling diverse biological processes in the developing endosperm. In particular, lack of LEC1 led to an altered endosperm metabolic phenotype in storage lipid content and fatty acid composition. Together, our findings provide important insights into the requirement of LEC1 for endosperm development.
Project description:This series contain all stages Arabidopsis plant development. Stages of development includes unfertilized ovule, 24-Hr post-fertilization seed, globular stage seed, cotyledon stage seed, mature green seed, post-mature green seed, post-germination seedling, rosette leaf, root, stem, and floral bud.
Project description:In plants, fatty acids are de novo synthesized predominantly in plastids fromacetyl-CoA. Although fatty acid biosynthesis has been biochemically well-studied, little isknown about the regulatory mechanisms of the pathway. Here, we show that overexpressionof the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causesglobally increased expression of fatty acid biosynthetic genes, which are involved in keyreactions of condensation, chain elongation and desaturation of fatty acid biosynthesis. Inthe plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes areupregulated in LEC1-overexpressing transgenic plants, including those encoding threesubunits of acetyl-CoA carboxylase, a key enzyme controlling the fatty acid biosynthesisflux. Moreover, genes involved in glycolysis and lipid accumulation are also upregulated.Consistent with these results, levels of major fatty acid species and lipids were substantiallyincreased in the transgenic plants. Genetic analysis indicates that the LEC1 function ispartially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3 and WRINKLED1 in theregulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed intransgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus.These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate theexpression of fatty acid biosynthetic genes, thereby representing a promising target forgenetic improvement of oil-production plants.
Project description:LEAFY COTYLEDON1 (LEC1), an atypical subunit of the NF-Y CCAAT binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene network and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. In this part of the study, we identified the genes bound by LEC1 in Arabidopsis bent cotyledon stage seeds. We expressed a GFP-tagged form of LEC1 under the control of its native promoter and terminator in lec1 null mutant seeds. Using an anti-GFP antibody (NeuroMab 75-131) , we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using chromatin isolated from bent cotyledon stage seeds to identify the genes bound by LEC1.
Project description:LEAFY COTYLEDON1 (LEC1), an atypical subunit of the NF-Y CCAAT binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene network and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. In this part of the study, we identified the genes regulated by LEC1 by overexpressing an inducible form of LEC1 in Arabidopsis seedlings and identifying the mRNAs induced EARLY (1h) after induction of LEC1 activity.
Project description:The transcription factor (TF) LEAFY COTYLEDON1 (LEC1) acts as an essential regulator of Arabidopsis embryogenesis and seed development. It controls aspects of early embryogenesis such as cotyledon identity and suspensor morphology, as well as seed maturation processes such as storage compound accumulation, acquisition of desiccation tolerance and dormancy. To identify downstream components of the LEC1 regulon, dexamethasone-regulated expression of LEC1 and ChIP/chip were applied and revealed the enrichment of phytohormone- as well as elongation-related genes among LEC1 target genes.
Project description:LEAFY COTYLEDON1 (LEC1), an atypical subunit of the NF-Y CCAAT binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene network and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. In this part of the study, we identified the genes bound by LEC1 by overexpressing a FLAG-tagged inducible form of LEC1 in Arabidopsis seedlings and inducing LEC1 activity for a short (4 hours) or long (8 days) period of time, to identify the genes bound EARLY and LATE, respectively.
Project description:LEAFY COTYLEDON1 (LEC1), an atypical subunit of the NF-Y CCAAT binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene network and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. In order to identify the genes bound by LEC1 in soybean embryos, we used two antibodies specific for soybean LEC1 (GmLEC1) and performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using chromatin isolated from soybean embryos at the cotyledon, early maturation and mid-maturation stages.