Chromatin immunoprecipitation of MADS29 to find the cis regulatory regions and targets of this transcription factor
Ontology highlight
ABSTRACT: A ChIP-seq analysis revealed 378 targets of OsMADS29, which include genes involved in cytokinin metabolism and auxin signaling, carbohydrate metabolism, transporters and a large number of transcription factors, reflecting on its functional diversity. Chromatin Immunonoprecipitation of MADS29 using antiMADS29 antibody was done using rice 9 DAP seed tissue with total input chromatin and mock immunoprecipitated chromatin as controls
Project description:To dissect the molecular mechanisms underlying drought tolerance (DT) in rice, transcriptome differences of a DT introgression line H471, the DT donor P28 and the drought sensitive recurrent parent HHZ under drought stress were investigated using deep transcriptome sequencing. Results revealed a differential constitutive gene expression prior to stress and distinct global transcriptome reprogramming among three genotypes under time-series drought stress, consistent with their differential genotypes and DT phenotypes. DT introgression line H471, the DT donor P28 and the drought sensitive recurrent parent HHZ under drought stress were investigated using deep transcriptome sequencing.The drought stress treatment was started by withholding water at the tillering stage. The days were counted after the AWC in the soil reached 20% to allow drought measurements at precisely determined intervals, and the soil water content reached 15%, 10% and 7.5% after 1d, 3d and 4d drought treatment, respectively.Three top leaves for each sample were harvested for each genotype under 1d and 3d drought stress and control conditions. All samples were immediately frozen in liquid nitrogen and stored at -80C and then for transcriptome sequencing.
Project description:The Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of Lysine 27 of Histone H3. The functions of the plant PRC2 have been chiefly described in Arabidopsis but specific PRC2 functions in other plant species, especially the cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis PRC2 gene EMBRYONIC FLOWER2 (EMF2). Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss of function mutants in rice E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 that are involved in floral development as differentially expressed in the emf2b mutant compared to wild type. OsMADS1 and OsMADS6 are known to be required for meristem determinacy in rice, and are down-regulated in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was up-regulated. Chromatin immunoprecipitation for H3K27me3 followed by deep sequencing showed that all three genes are amongst the presumptive targets of PRC2 in the meristem. We propose that the PRC2 operates through a mechanism that involves regulation of E-function genes to play a major role in floral organ specification and floral meristem determinacy in rice, and possibly in other cereals. RNA-seq: The transcriptome of WT and emf2b rice panicles were compared via RNA-seq.
Project description:Nucleosome free measurement of 14 day old rice leaves (2nd leaf) in heat stress and recovery and dehydration stress and recovery 5 conditions: control (30C, liquid media; at 0.5h, 2h, 4h); Heat (transferred from 30C to 40C; at 0.5h, 2h, 4h); Heat recovery (transferred back to 30C after 2h at 40C; after 2h); Dehydration (roots exposed to air; at 2h); Dehydration recovery (roots returned to liquid media after 1.5h in air; after 2h) Samples: 2 biological replicates.
Project description:The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, while the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells, and pollen vegetative cells from rice, and identified transcripts for ~36,000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions and their contributions to zygote and seed development. The gene expression profiles of the three gametic cells, egg cell (EC), sperm cell (Sp) and vegetative cell (Ve) were compared via RNA-seq using three biological replicates for each. Differential expression (DE) analysis was performed using edgeR and the resulting DE genes were assessed for Gene Ontology term enrichment.
Project description:Illumina sequencing was employed to examine the expression profiles of rice anther miRNAs from the a non-pollen male sterile line Wuxiang S (WXS), one of photo-thermo sensitive genical male sterile (PTGMS) line rice, during in the fertility transition stage. A total of 493 known miRNAs and 273 novel miRNAs were identified during rice anther development. Based on the number of sequencing reads, a total of 26 miRNAs were discovered to be significant difference expression between WXS(S, Sterility) and WXS(F, Fertility), and the results were partially validated by qRT-PCR. Among these, 11 miRNAs were decreased and 15 miRNAs were increased in WXS(S) compared with WXS(F). The expression patterns for targets of osa-miR156a-j, osa-miR3879, osa-miR159c/d/e, osa-miR171a/c/e/i, osa-miR398b, osa-miR164d, osa-miR528 and osa-miR408 were selectively examined, and the results showed that there was a negative correlation on the expression patterns between miRNAs and their targets. These targets have previously been reported to be related with pollen development and male sterility, suggesting that miRNAs might act as regulators of rice anthers. Furthermore, miRNA editing events were observed. The U-to-C and U-to-A editing phenomenon was validated by molecular cloning and sequencing. Examine small RNA profiles change of four tissues of the rice non-pollen male sterile line Wuxiang S under two different environments.
Project description:RNA-Seq was performed to study the change of gene expression before and after chilling treatment in rice. Different change patterns were identified. We have provided a complete view of transcriptome under cold stress condition, which will deepen our understanding of gene expression regulation in cold stress response as well as cold stress response mechanism for monocot plants. The mRNA profiles of 15-day-old rice seedlings with and without chilling treatment (4 °C for 33 h) were generated by deep sequencing using Illumina Hiseq 2000 platform.
Project description:The members of bHLH transcription factor superfamily are known to play key role in plant development and abiotic stress response. Loss-of-function of OsbHLH148 gene resulted in increased sensitivity of rice plants to drought stress. To identify the targets of OsbHLH148 and dissect the drought stress response pathway regulated by it, we performed transcriptome profiling of Osbhlh148 mutant plants under drought stress as well as well-watered conditions by RNA-sequencing. OsbHLH148 loss-of-function rice plants (Oryza sativa ssp. japonica cv. Nipponbare) were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.
Project description:In this study, genome-wide transcriptome profiling was used to understand molecular genetic mechanism of drought tolerance in rice. Illumina High-Seq 2000 platform was used for sequencing RNA from leaf tissue of rice plants exposed to controlled drought stress and well-watered conditions. The differentially expressed genes were used to identify biological process and cis-regulatory elements enriched under drought stress compared to well-watered conditions. Oryza sativa ssp. japonica cv. Nipponbare plants were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.
Project description:Heat shock factors (Hsfs) are known to regulate heat and drought stress response by controlling the expression of heat shock proteins and oxidative stress responsive genes. Loss-of-function of OsHSFA2e gene resulted in increased sensitivity of rice plants to drought and heat stress. To identify the targets of OsHSFA2e and dissect the stress response pathway regulated by it, we performed transcriptome profiling of Oshsfa2e mutant plants under drought stress as well as well-watered conditions by RNA-sequencing. OsHSFA2e loss-of-function rice plants (Oryza sativa ssp. japonica cv. Nipponbare) were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.