Project description:The capacity for achieving immense specificity and resolution in science increases day to day. Fluorescence-activated nuclear sorting (FANS) offers this great precision, enabling one to count and separate distinct types of nuclei from specific cells of heterogeneous mixtures. We developed a workflow to collect nuclei from Arabidopsis thaliana by FANS according to cell lineage and endopolyploidy level with high efficiency. We sorted GFP-labeled nuclei with different ploidy levels from the epidermal tissue layer of three-day, dark-grown hypocotyls followed by a shift to light for one day and compared them to plants left in the dark. We then accessed early chromatin accessibility patterns associated with skotomorphogenesis and photomorphogenesis by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) within primarily stomatal 2C and fully endoreduplicated 16C nuclei. Our quantitative analysis shows that dark- and light-treated samples in 2C nuclei do not exhibit any different chromatin accessibility landscapes, whereas changes in 16C can be linked to transcriptional changes involved in light response.
Project description:We produced RNA-seq reads from messenger RNA isolated from aerial seedling tissue for Arabidopsis thaliana mutants in the HULK gene family. The read data were generated with biological replication (two replicates). The resulting RNA-seq data provide a resource to assess the function of HULK genes in the control of downstream gene expression in A. thaliana. Examination of RNA transcript levels in HULK T-DNA mutants (and mutant combinations therein) in Arabidopsis thaliana.
Project description:BACKGROUND:Regulation of transcription depends on interactions between cis-regulatory elements (CREs) and regulatory proteins. Active CREs are imbedded in open chromatin that are accessible to nucleases. Several techniques, including DNase-seq, which is based on nuclease DNase I, and ATAC-seq, which is based on transposase Tn5, have been widely used to identify genomic regions associated with open chromatin. These techniques have played a key role in dissecting the regulatory networks in gene expression in both animal and plant species. RESULTS:We develop a technique, named MNase hypersensitivity sequencing (MH-seq), to identify genomic regions associated with open chromatin in Arabidopsis thaliana. Genomic regions enriched with MH-seq reads are referred as MNase hypersensitive sites (MHSs). MHSs overlap with the majority (~?90%) of the open chromatin identified previously by DNase-seq and ATAC-seq. Surprisingly, 22% MHSs are not covered by DNase-seq or ATAC-seq reads, which are referred to "specific MHSs" (sMHSs). sMHSs tend to be located away from promoters, and a substantial portion of sMHSs are derived from transposable elements. Most interestingly, genomic regions containing sMHSs are enriched with epigenetic marks, including H3K27me3 and DNA methylation. In addition, sMHSs show a number of distinct characteristics including association with transcriptional repressors. Thus, sMHSs span distinct classes of open chromatin that may not be accessible to DNase I or Tn5. We hypothesize that the small size of the MNase enzyme relative to DNase I or Tn5 allows its access to relatively more condensed chromatin domains. CONCLUSION:MNase can be used to identify open chromatin regions that are not accessible to DNase I or Tn5. Thus, MH-seq provides an important tool to identify and catalog all classes of open chromatin in plants.
Project description:Light, a dynamic environmental parameter, is an essential regulator of plant growth and development. Light-regulated transcriptional networks are well documented, whereas light-regulated post-transcriptional regulation has received limited attention. In this study, dynamics in translation of cytosolic mRNAs were evaluated at the genome-level in Arabidopsis thaliana seedlings grown under a typical light/dark diurnal regime, shifted to darkness at midday, and then re-illuminated. One-hour of unanticipated darkness reduced levels of polysomes by 17% in a manner consistent with inhibition of initiation of translation. This down-regulation of translation was reversed within 10?min of re-illumination. Quantitative comparison of the total cellular population of transcripts (the transcriptome) to those associated with one or more 80S ribosome (the translatome) identified over 1600 mRNAs that were differentially translated in response to light availability. Unanticipated darkness limited both transcription and translation of mRNAs encoding components of the photosynthetic machinery. Many mRNAs encoding proteins associated with the energy demanding process of protein synthesis were stable but sequestered in the dark, in a rapidly reversible manner. A meta-analysis determined these same transcripts were similarly and coordinately regulated in response to changes in oxygen availability. The dark and hypoxia translationally repressed mRNAs lack highly supported candidate RNA-regulatory elements but are characterized by G?+?C-rich 5'-untranslated regions. We propose that modulation of translation of a subset of cellular mRNAs functions as an energy conservation mechanism.
Project description:Background:There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever-growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors (TFs) to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus, the accessibility of these loci for TF binding is highly regulated and determines where and when TFs bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana and define organ-specific regulatory sites and binding motifs of TFs at these sites. Results:We coupled the recently described isolation of nuclei tagged in specific cell types (INTACT) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions but are correlated with root-specific expression of distant genes. Importantly, these gene-distant sites are enriched for binding motifs of TFs important for root development as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel gene-distant regulatory elements. Conclusions:By coupling INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root-specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.
Project description:We mapped DNaseI hypersensitive sites (DHSs) and applied genomic footprinting to define in vivo transcription factor (TF) occupancy at nucleotide resolution across the A. thaliana genome in whole seedlings during heat- and light-response states. We find that trait-associated variation localizes within DHSs, and that extensive TF occupancy within protein-coding exons has shaped A. thaliana codon usage. Analysis of >700,000 TF footprints disclosed an extensive cis-regulatory lexicon, and enabled construction of large-scale TF cross-regulatory networks. Although the cis- and trans-regulatory repertoire is markedly distinct from mammals, the architecture of A. thaliana TF networks is strikingly similar to those of human. Analysis of the DHS landscape and TF network dynamics during heat shock and photomorphogenesis revealed thousands of conditionally-sensitive elements and enabled mapping of key regulatory circuits. Our results provide an extensive resource for understanding and enabling diverse aspects of A. thaliana biology. Chromatin accessibility profiling (Dnase I-seq) and RNA-seq of 7-day-old dark-grown seedlings exposed to 0hr light, 30min light, 3hr light or 24hr LD conditions. Chromatin accessibility profiling (Dnase I-seq) and RNA-seq of 7-day-old LD-grown seedlings treated with a brief sever heat shock or kept under control conditions. Replicates are included when available; controls and read-normalized samples (samples that were subsampled to the same read-depth) are also included here.
Project description:Arabidopsis thaliana and Arabidopsis lyrata are two closely related Brassicaceae species, which are used as models for plant comparative biology. They differ by lifestyle, predominant mating strategy, ecological niches and genome organization. In order to explore molecular basis of specific traits, we performed RNA-sequencing of vegetative rosettes from both species. Additionally, we sequenced apical meristems and inflorescences of A. lyrata that allow for intra-specific transcriptome comparison in several major developmental stages. Arabidopsis lyrata and Arabidopsis thaliana aerial tissues were collected from mock treated plants, total RNA isolated and poly-A RNA populations sequenced
Project description:Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development.
Project description:Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.
Project description:Plant circadian systems are composed of a large number of self-sustained cellular circadian oscillators. Although the light-dark signal in the natural environment is known to be the most powerful Zeitgeber for the entrainment of cellular oscillators, its effect is too strong to control the plant rhythm into various forms of synchrony. Here, we show that the application of pulse perturbations, i.e., short-term injections of darkness under constant light, provides a novel technique for controlling the synchronized behavior of plant rhythm in Arabidopsis thaliana. By destroying the synchronized cellular activities, circadian singularity was experimentally induced. The present technique is based upon the theory of phase oscillators, which does not require prior knowledge of the detailed dynamics of the plant system but only knowledge of its phase and amplitude responses to the pulse perturbation. Our approach can be applied to diverse problems of controlling biological rhythms in living systems.