Transcriptional profiles of Arabidopsis stomataless mutants
Ontology highlight
ABSTRACT: Stomataless Arabidopsis thaliana mutants spch-3 and mute-3 are extreme dwarfs but can produce cotyledons and tiny leaves, providing a system to interrogate plant life in the absence of stomata. To this end, we compared the transcriptomes of mutant cotyledons lacking SPCH or MUTE functions, and therefore lacking stomata, with those of wild-type, stomata-bearing Col-0 accession. RNA was obtained from 21 days old cotyledons collected from 9 independent biological replicates per genotype. These were pooled in 3x3 replicates and used to hybridize Affymetrix ATH1 microarrays.
Project description:Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposon mobility during plant developmental processes. Recently, the variation of DNA methylation has been described between wild type and DNA methylation-related mutants in Arabidopsis thaliana. However, the elaborate representation of soybean DNA methylomes remains lacking. Here, we described the epigenome maps of soybean root, stem, leaf, and cotyledon of developing seed at a single-base resolution. We confirmed the transcription start sites of genes using high-throughput sequencing and reported the DNA methylation patterns in gene and transposon regions. The correlation between gene expression and DNA methylation was revealed through transcriptome sequencing. We found CHH methylation may function in promotion of gene expression and ten cotyledon-preferred genes were identified CHH hypermethylated in cotyledon. Small RNA library sequencing showed that DNA methylation was enhanced by small RNAs not by strand-specific way, and the variation of DNA methylation between the organs was highly related with expression of small RNAs. methylomes of roots, stems, leaves, and cotyledons of developing seeds
Project description:We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype. In order to compare the population of chalcone synthase related small RNAs, we sequenced 3 to 6 million small RNAs using the Illumina Genome Analyzer from the following four soybean cultivars and tissues with specific genotypes at the I locus: Richland immature seed coats (homozygous for the dominant I allele that specifies yellow seed coat); Williams immature seed coats (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum) Williams (i-i/i-i yellow) immature cotyledons (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum); Williams 55 immature seed coats (a Williams isogenic line homozygous for the recessive i allele that specifics pigmented seed coats. All seed coats and cotyledons were dissected from green stage immature seeds within the fresh weight range of 50-75 mg.
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants.
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants.
Project description:Five degradome libraries were constructed from three different seed developmental stages. Separate degradome libraries were constructed for seed coat and cotyledons to identify the tissue specific miRNAs and their potential targets. Sequencing and analysis of degradome libraries gives identification of 183 different targets for 80 known soybean miRNAs. We found 30 cotyledon specific, 18 seed coat specific and 32 miRNAs found in both tissues irrespective of the developmental stages. One interesting observation is that we found more miRNA targets in late seed developmental stages than earlier stages. Additionally, we have validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5? rapid amplification of cDNA ends (RLM-5?RACE). GO analysis indicated the enrichment of miRNA target genes in seed development. Construction of degradome libraries using cotyledons and seed coats from 3 different developmental stages
Project description:Virus resistances that are recessively inherited are associated with loss-of-susceptibility resistance alleles. Resistance to Watermelon mosaic virus (WMV) of melon accession TGR-1551 is expressed as a drastic reduction of the virus titer, and is recessively inherited. In this work, viral RNA accumulation was measured in TGR-1551 and in susceptible WMV-infected melon plants by real time quantitative PCR (qPCR), and gene expression of 17,443 unigenes represented in a melon microarray was monitored in a time-course experiment. Virus accumulation was higher in inoculated cotyledons of the resistant genotype up to 7 days post-inoculation; from this time on, virus accumulation was much higher in plants of the susceptible genotype. Microarray experiments were carried with samples from inoculated cotyledons at 1 and 3 dpi to monitor early changes in response to virus infection, and at 7 dpi. Samples from systemically infected leaves harvested at 15 dpi were also included in the analysis. Results showed much more profound transcriptomic alterations in resistant plants compared to susceptible ones. Analyses of gene expression profiles reveal deep and extensive transcriptomic alterations in TGR-1551 plants, many of them involving pathogen response-related genes. Overall, data suggested that resistance to WMV in TGR-1551 is associated with a defense response, contrasting with its recessive nature. Two melon genotypes have been used to analyse transcriptomic responses to infection by Watermelon mosaic virus: Tendral (susceptibel to WMV) and TGR-1551 (resistant to WMV). For each genotype, 60 melon seedlings were inoculated with WMV-M116 and another 60 were mock-inoculated. Cotyledons of 10 plants were harvested at 1, 3, 5, 7, 9 and 15 dpi. At 15 dpi, the systemically infected second true leaf was also harvested. To reduce variability, each biological replicate used in this study was prepared by mixing the RNA extracts from 2 or 4 mock or WMV-inoculated cotyledons, respectively, or from 3 melon leaves. Samples (WMV infected and mock inoculated) corresponding to cotyledons at 1, 3 and 7 dpi, and leaves at 15 dpi were used for microarray hybridisations, three biological replicates for each one, leading to a total of 48 samples.
Project description:How epigenetics is involved in the transition from seed maturation to seed germination largely remains elusive. To uncover the possible role of epigenetics in gene expression during the transition from seed maturation to seed germination in soybean, the transcriptome of cotyledons from four stages of soybean seed maturation and germination, including mid-late maturation, late maturation, seed dormancy and seed germination, were profiled by Illumina sequencing. For the genes that are quantitatively regulated at the four stages, two antagonistic epigenetic marks, H3K4me3 and H3K27me3, together with the binding of RNA polymerase II, were investigated at the four stages by chromatin immunoprecipitation (ChIP). For 10 out of 16 genes examined, the relative enrichment of histone modification marks (H3K4me3 and H3K27me3) and RNA polymerase II binding on their promoter regions correlates well with their relative expression levels at four stages, suggesting the involvement of epigenetics in transcriptional regulation. A striking finding is that seed germination-specific genes start to show open chromatin (H3K4me3) during late seed maturation although their transcripts do not accumulate, which is further supported by RNA polymerase II binding. Together, our results provide the first evidence that seed germination genes can be primed for transcription (open chromatin and RNA polymerase II binding) during seed maturation, highlighting that the transition from seed maturation to seed germination starts at late seed maturation stages at both the genetic and epigenetic levels. Illumina sequencing of transcripts from cotyledons of mid-maturation (B1 stage) and late maturation (AA1 stage) seeds, whole dry seeds, and cotyledons of seedlings six days after imbibition.
Project description:Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposon mobility during plant developmental processes. Recently, the variation of DNA methylation has been described between wild type and DNA methylation-related mutants in Arabidopsis thaliana. However, the elaborate representation of soybean DNA methylomes remains lacking. Here, we described the epigenome maps of soybean root, stem, leaf, and cotyledon of developing seed at a single-base resolution. We confirmed the transcription start sites of genes using high-throughput sequencing and reported the DNA methylation patterns in gene and transposon regions. The correlation between gene expression and DNA methylation was revealed through transcriptome sequencing. We found CHH methylation may function in promotion of gene expression and ten cotyledon-preferred genes were identified CHH hypermethylated in cotyledon. Small RNA library sequencing showed that DNA methylation was enhanced by small RNAs not by strand-specific way, and the variation of DNA methylation between the organs was highly related with expression of small RNAs. small RNA profiling of roots, stems, leaves, and cotyledons of developing seeds
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants. Genome-wide identification of SPCH binidng sites
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants. RNA-Seq profiles of inducible SPCH and wild-type upon induction