Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of Arabidopsis thaliana siliques from WT and MYB73 overexpression lines

ABSTRACT: This experiment seeks to elucidate the functional role of MYB73 in Arabidopsis thaliana siliques via differential gene expression (DGE) analysis. Total RNA were extracted from pooled Arabidopsis siliques at 12 days after flowering (DAF) for 3 biological replicates from WT and MYB73-OE lines. RNA-seq libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) for Illumina according to the manufacturer’s instruction and sequenced with Novaseq-6000 (Illumina) using paired-end sequencing with read lengths of 150 base pairs at sequencing depths of ~ 2 million reads per sample.

INSTRUMENT(S): Illumina NovaSeq 6000, Kallisto

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Marek Mutwil

PROVIDER: E-MTAB-14541 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology and defective hormonal and stimulus responses. HY5 thus acts as an integrater that links various gene networks to coordinate plant development. Here we report an effort to experimentally map the HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate HY5 binds to over 9,000 genes, which detectably impact the expression of over 1,100 genes, either positively or negatively. Further, HY5 indirectly regulates many other genes through sub-networks mediated by other regulators. In particular, we show that HY5 regulates eight microRNA (miRNA) genes, which in turn control transcript abundance of specific target genes. Over-expressing the HY5-targeted miR408 resulted in phenotypes that are opposite to hy5 mutants. Together our results revealed both the transcriptional and post-transcriptional components of the HY5-mediated gene networks responsible for the phenotypic complexity in plants. DNase I-treated total RNA was prepared from wild type and hy5-215 seedlings grown under wild type light (WL). The mRNA fraction was extracted from total RNA using Dynabeads oligo(dT)25 (Invitrogen) following the manufacturerM-bM-^@M-^Ys directions. First- and second-strand cDNA were generated usingSuperScript II and random hexamer primers. Double-stranded cDNA was fragmented by nebulization and used for mRNA library construction following the manufacturerM-bM-^@M-^Ys protocol (Illumina). Bowtie (Langmead et al., 2009) was used to map the sequence reads to the Arabidopsis genome and only uniquely mapped reads were used in subsequent analyses.

Project description:Background: The carpel margin meristem is a vital multi-potent structure located in the medial domain of the Arabidopsis thaliana gynoecium, the female floral reproductive organ. The carpel margin meristem generates ovules that upon fertilization become seeds. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present an analysis of the transcriptional profile of the medial domain of the Arabidopsis gynoecium highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Results: Using a floral synchronization system and a SHATTERPROOF2 domain-specific reporter construct, paired with fluorescence-activated cell sorting, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. Our analysis reveals a set of genes that are differentially-expressed within the SHATTERPROOF2 expression domain that marks portions of the developing medial domain. Many members of this gene set have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Other uncharacterized members of this gene set, including a set of differentially-expressed cis-natural antisense transcripts, are potential novel regulators of medial domain development and candidates for future functional studies. Several members of the REM family of transcriptional regulators were enriched in the SHP2-expressing cell population including a previously unrecognized REM family member (At5g60142). Analysis of the abundance of specific transcriptional isoforms identified genes that may exhibit âisoform switchingâ behavior during gynoecial development. Conclusions: This data set provides genome-wide transcriptional insight into the development of the gynoecial medial domain that contains the carpel margin meristem, an important reproductive structure that gives rise to the ovules in Arabidopsis thaliana. Four samples (YFP-positive, YFP-negative, all-sorted, and non-sorted). Four biological replicates of each sample (except YFP-positive, collected in triplicated). Strand-specific libraries were sequenced (single-end) in two lanes of a HiSeq 2500 Illumina flow cell (each lane was later analyzed as a technical replicate).

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RNA-seq of Arabidopsis thaliana siliques from WT and MYB73 overexpression lines

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