Identification of LEC1 binding sites in Arabidopsis seeds during embryo expansion stage
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ABSTRACT:
PROVIDER: PRJNA432504 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6661123.fastq.gz | Fastqsanger.gz | |||
| SRR6661124.fastq.gz | Fastqsanger.gz | |||
| SRR6661125.fastq.gz | Fastqsanger.gz | |||
| SRR6661126.fastq.gz | Fastqsanger.gz |
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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.
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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.
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Project description:To understand how GA functions in regulating embryo development, a genome-wide transcriptomic analysis was carried out using 9DAF seeds dissected from siliques of dellaq (rga28 gai rgl1 rgl2) and the wild-type (col-0-2) grown in full-spectrum white fluorescent light at 22°C under long day conditions (16 h light/8 h dark). Then we found that GA regulates embryo development via DELLA-LEC1interaction, a subsequent genome-wide transcriptomic analysis was carried out using 9DAF seeds dissected from siliques of lec1-4 and the wild-type (col-0-1) in the same growth condition. Basing on the criteria of 1.5-fold cutoff for the genes with 5% false discovery rate, we first identified the differentially expressed genes in dellaq vs col-0-2, lec1-4 vs Col-0-1 subsets, which are referred to as DELLA and LEC1 regulated genes. These data reveal that DELLAs and LEC1 co-target a set of common genes in late embryogenesis, strongly supporting the role of DELLA-LEC1 in embryo development.
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Project description:We collected globular stage seed compartments from 5 or 7-micron paraffin sections using the Leica LMD6000 system in order to identify the mRNAs present in different compartments of an Arabidopsis seed containing a globular stage embryo. For the purposes of this study we broke down the seed into 8 capturable compartments: embyro proper, suspensor, micropylar endosperm, peripheral endosperm, chalazal endosperm, chalazal seed coat, general seed coat, and whole seeds. Keywords: cell type comparison
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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.
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Project description:In species with exalbuminous seeds such as crucifer oilseeds and legumes, the endosperm is eventually consumed and its space occupied by the embryo during seed development. However, the main constituent of the early developing seed is the liquid endosperm, and most of the carbon resources for the ensuing stages of seed development arrive at the embryo through the endosperm. In contrast to the extensive study of species with persistent endosperm, little is known about the global gene expression pattern in the endosperm of exalbuminous seed species. We took a multiparallel approach that combines ESTs, protein profiling and microarray analyses to look into the gene expression landscape in the endosperm of the oilseed crop Brassica napus. An EST collection of over 30,000 entries allowed us to detect close to 10,000 unisequences expressed in the endosperm. A protein profile analysis of more than 800 proteins corroborated several signature pathways uncovered by abundant ESTs. Using microarray analyses, we identified genes that are differentially or highly expressed across all developmental stages. These complementary analyses provided insight on several prominent metabolic pathways in the endosperm. We also discovered that LEC1 was highly expressed in the endosperm and that the regulatory cascade downstream of LEC1 operates in the endosperm. The endosperm EST collection and the microarray dataset provide a basic genomic resource for dissecting metabolic and developmental events important for oilseed improvement. Our findings on the featured metabolic processes and the LEC1 regulatory cascade offer new angles for investigation on the integration of endosperm gene expression with embryo development and storage product deposition in seed development. Keywords: seed development We defined the developmental phase of the endosperm based on the stages of the embedded embryos. Under our growth conditions, the globular-shape embryo stage is reached approximately six days after flowering (DAF), the heart-shape embryo stage is reached after eight days, and by day 14 the seed enters the cotyledon stage. The collected tissues allowed the construction of two cDNA libraries: one derived from endosperm of heart-shape embryo developing stage seeds and the other from endosperm of a mix of globular-shape embryo and cotyledon stage developing seeds. Heart-shaped-embryo endosperm is also ready for protein profiling analysis. For microarray analysis, endosperms were harvested from young seeds at the globular-shape embryo, heart-shape embryo and cotyledon stages, respectively. Two biological duplicates were set up, including four technological repeats in each duplicate. A dye-swap hybridization experiment was performed for each pair of target RNA comparisons. We performed four independent aRNA labelings for each tissue pair. For example, we used globular-shape embryo stage endosperm RNA coupled with Cy5 dye vs. heart-shape embryo stage endosperm RNA coupled with Cy3 dye, and the dye-swap experiment was with globular-shape embryo stage endosperm RNA with Cy3 dye vs. the heart-shape embryo stage endosperm RNA with Cy5 dye. Each tissue pair experiment was done with four repeats, using a total of 24 microarrays for the whole experiment.
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