Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

SEEDSTICK Is a Master Regulator of Development and Metabolism in Arabidopsis Seed Coat

ABSTRACT: Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from the SEEDSTICK mutant in Arabidopsis with a wild type, to unveil the role of this transcription factor in seed development and decipher the impact of this factor in PAs metabolism Methods: Total RNA was extracted from two biological replicates from both wild-type and stk mutant inflorescences and siliques until 5 DAP with the Qiagen Kit according to the manufacturer's instructions. DNA contaminations were removed using the PROMEGA RQ1 RNase-Free DNase according to the manufacturer's instructions. RNA quality integrity was analyzed by electrophoresis gel and was validated on a Bioanalyzer 2100 (Aligent, Santa Clara, CA); RNA Integrity Number (RIN) values were greater than 7 for all the samples. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5â??-TGCGATGCAGAAGTTGCGCTC-3â??) and RT 781 (5â??-AGTACGCGGCATTGATTTCTTG-3â??). Sequencing libraries were prepared according to the manufacturerâ??s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. The processing of fluorescent images into sequences, base-calling and quality value calculations were performed using the Illumina data processing pipeline (version 1.8). Raw reads were filtered to obtain high-quality reads by removing low-quality reads containing more than 30% bases with Q < 20. Finally, a quality control of the raw sequence data was performed using FastQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Results: A total of 102,278,242 reads passed a quality filter and 85% were mapped back to the Arabidopsis TAIR10 genome. Approximately 90% mapped uniquely to only one location and could be assigned to a single annotated TAIR10 gene. Normalization of expression values was performed using RPKM values. All other parameters were kept at default levels. The CLC Genomic Workbench was also further used to determine all differentially expressed transcripts found in each cDNA library. Baggerley's test and a FDR correction were used for statistical analysis of samples. Our analysis revealed that 156 genes were upregulated, whereas for 90 genes a reduction in their mRNA level was observed in the stk mutant when compared to wild-type . Data analysis revealed a significant enrichment for terms related to the phenylpropanoid metabolic process, flavonoid biosynthetic process as well as cellular amino acid derivative metabolic process. Conclusion: Our genome-wide transcriptomic analysis suggests that the ovule identity factor STK is involved in the regulation of several metabolic processes providing a strong connection between cell fate determination, development and metabolism. In particular we characterize, through phenotypic, genetic, biochemical and transcriptomic approaches, the role of STK in PAs biosynthesis. Our results indicate that STK exerts this role through the direct regulation of the gene encoding for BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This supports the theory that MADS-domain proteins control the expression of their target genes through the modification of the chromatin states. STK might recruit or negatively regulate histone modifying factors to control their activity. Moreover, we show that STK controls through a complex regulatory network not only directly BAN but also other regulators of this key gene in tannin production mRNA profiles from both Arabidopsis wild-type and stk mutant inflorescences and siliques until 5 DAP were generated by deep sequencing, in duplicate according to the manufacturerâ??s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5â??-TGCGATGCAGAAGTTGCGCTC-3â??) and RT 781 (5â??-AGTACGCGGCATTGATTTCTTG-3â??).

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: IGNACIO EZQUER

PROVIDER: E-GEOD-59637 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat.

Mizzotti Chiara C Ezquer Ignacio I Paolo Dario D Rueda-Romero Paloma P Guerra Rosalinda Fiorella RF Battaglia Raffaella R Rogachev Ilana I Aharoni Asaph A Kater Martin M MM Caporali Elisabetta E Colombo Lucia L

PLoS genetics 20141218 12

The role of secondary metabolites in the determination of cell identity has been an area of particular interest over recent years, and studies strongly indicate a connection between cell fate and the regulation of enzymes involved in secondary metabolism. In Arabidopsis thaliana, the maternally derived seed coat plays pivotal roles in both the protection of the developing embryo and the first steps of germination. In this regard, a characteristic feature of seed coat development is the accumulat ...[more]

PMID: 25521508

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Project description:Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from the SEEDSTICK mutant in Arabidopsis with a wild type, to unveil the role of this transcription factor in seed development and decipher the impact of this factor in PAs metabolism Methods: Total RNA was extracted from two biological replicates from both wild-type and stk mutant inflorescences and siliques until 5 DAP with the Qiagen Kit according to the manufacturer's instructions. DNA contaminations were removed using the PROMEGA RQ1 RNase-Free DNase according to the manufacturer's instructions. RNA quality integrity was analyzed by electrophoresis gel and was validated on a Bioanalyzer 2100 (Aligent, Santa Clara, CA); RNA Integrity Number (RIN) values were greater than 7 for all the samples. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5’-TGCGATGCAGAAGTTGCGCTC-3’) and RT 781 (5’-AGTACGCGGCATTGATTTCTTG-3’). Sequencing libraries were prepared according to the manufacturer’s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. The processing of fluorescent images into sequences, base-calling and quality value calculations were performed using the Illumina data processing pipeline (version 1.8). Raw reads were filtered to obtain high-quality reads by removing low-quality reads containing more than 30% bases with Q < 20. Finally, a quality control of the raw sequence data was performed using FastQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Results: A total of 102,278,242 reads passed a quality filter and 85% were mapped back to the Arabidopsis TAIR10 genome. Approximately 90% mapped uniquely to only one location and could be assigned to a single annotated TAIR10 gene. Normalization of expression values was performed using RPKM values. All other parameters were kept at default levels. The CLC Genomic Workbench was also further used to determine all differentially expressed transcripts found in each cDNA library. Baggerley's test and a FDR correction were used for statistical analysis of samples. Our analysis revealed that 156 genes were upregulated, whereas for 90 genes a reduction in their mRNA level was observed in the stk mutant when compared to wild-type . Data analysis revealed a significant enrichment for terms related to the phenylpropanoid metabolic process, flavonoid biosynthetic process as well as cellular amino acid derivative metabolic process. Conclusion: Our genome-wide transcriptomic analysis suggests that the ovule identity factor STK is involved in the regulation of several metabolic processes providing a strong connection between cell fate determination, development and metabolism. In particular we characterize, through phenotypic, genetic, biochemical and transcriptomic approaches, the role of STK in PAs biosynthesis. Our results indicate that STK exerts this role through the direct regulation of the gene encoding for BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This supports the theory that MADS-domain proteins control the expression of their target genes through the modification of the chromatin states. STK might recruit or negatively regulate histone modifying factors to control their activity. Moreover, we show that STK controls through a complex regulatory network not only directly BAN but also other regulators of this key gene in tannin production

Project description:MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We now demonstrate that endogenous and over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). We report that the "seed region" of moR-21 as well as the "seed match region" in the target gene 3'UTR are indispensable for moR-21-mediated gene down-regulation. We further demonstrated that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. Taken together, these findings provide the first evidence that microRNA offset RNA regulates gene expression and is biologically active. Small RNAs were harvested from mouse aortic smooth muscle cells (AoSMCs), with three biological replicates, using miReasy kits (Qiagen) according to the manufacturerâ??s instructions. RNA was quantified using a Nanodrop spectrophotometer. Small RNA library construction and illumina HiSeq 75bp single read sequencing were performed at the Yale Center for Genome Analysis. Reads were trimmed to remove adaptor sequences, and inserts longer than 13 bases were mapped to the mouse mm10 genome using Bowtie (with settings â??n 0 -m 5 --best --strata, allowing 0 mismatches and up to 5 genomic loci with a perfect match, {Genome Biol. 2009;10(3):R25, Langmead B, Trapnell C, Pop M, Salzberg SL}). Reads that mapped to within 50 bp of more than one annotated mouse pri-miR (as defined by miRBase {Kozomara A, Griffiths-Jones S. Nucelic Acids Res 2014 42:D68-D73}), were allotted evenly between these locations. Reads that fell entirely within established miRbase 3p or 5p miR coordinates, plus or minus 3 bp, were counted as 3p or 5p miRs, respectively. Reads that fell entirely within the range of 35 bp 3â?? to 3 bp 5â?? of the last base of a 3p miR were counted as 3p moR reads. Those that fell entirely between 3bp 3â?? and 35 bp 5â?? of the first base of a 5p miR were counted as 5p moR reads. On average each library contained 17.4 million qualifying inserts, of which 13.8 million (79.3%) mapped to one of these four regions, indicating that the large majority of inserts represented miR or moR sequences. Reads were divided by the number of million reads mapping to these regions in each library to give normalized reads per million reads (RPMR) values.

Dataset Information

SEEDSTICK Is a Master Regulator of Development and Metabolism in Arabidopsis Seed Coat

Publications

SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat.

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