Project description:RNA-Seq of Arabidopsis thaliana Col-0 and 35S::FLAG-GR-KAN1 plants grown in ambient light or shade. 8 samples (Col-0 wildtype, 35S::FLAG-GR-KAN1 each in shade or ambient light, each with mock or Dexamethasone treatment) with two technical replicates were sequenced.
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified.
Project description:A high-depth strand-specific RNA sequencing (ssRNA-seq) using 5-day-old white-light grown Col-0 and pif7-1 seedlings with additional 1 hr shade or white light treatment.
Project description:For shade-intolerant species, shade light indicates the close proximity of neighboring plants and triggers the shade avoidance syndrome (SAS), which causes exaggerated growth and reduced crop yield. We report that nonsecreted ROT FOUR LIKE (RTFL)/DEVIL (DVL) peptides negatively regulate the SAS by repressing the activities of BRASSINOSTEROID SIGNALING KINASEs (BSKs) and PHYTOCHROME INTERACTING FACTOR 4 (PIF4) in Arabidopsis. To identify RTFL function during SAS, we performed RNA-seq to search for differentially expressed genes (DEGs) by comparing transcript levels between Col-0 and dvl1-1D, bsk36, or pif47 seedlings during white light and shade conditions.
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified. 10-day-old 35S::FLAG-GR-rREV plants were treated with 6-benzylaminopurine (6-BA), dexamethasone (DEX), or 6-BA+DEX for 2 hours. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. Total RNA was extracted with RNeasy Mini Kit and hybridized to Agilent Arabidopsis Gene Expression Microarray. Differentially expressed genes were defined by a 1.5-fold expression difference with a P value<0.05. Biological replicates were performed.
Project description:One of the key innovations of the flowering plants is their female reproductive organ, the carpel. Here we show that a mechanism controlling carpel margin development in the model flowering plant Arabidopsis thaliana was recruited from light-regulated processes. This recruitment followed the loss from the basic Helix-Loop-Helix transcription factor SPATULA (SPT) of a domain previously responsible for its negative regulation by phytochrome. We propose that the loss of this domain was a prerequisite for the light-independent expression in female reproductive tissues of a genetic module that also promotes shade avoidance responses in vegetative organs. Striking evidence for this proposition is provided by the restoration of wild type carpel development to spt mutants by low red/far-red light ratios, simulating vegetation shade, which we show to occur via PHYB, PIF4 and PIF5. Our data illustrate the potential of modular evolutionary events to generate rapid morphological change, and thereby provide a molecular basis for neo-Darwinian theories that describe this non-gradualist phenomenon. Furthermore, the effects shown here of light quality perception on carpel development lead us to speculate on the potential role of light-regulated mechanisms in plant organs that, like the carpel, form within the shade of surrounding tissues. The SPATULA (SPT) coding sequence was fused to the 5M-bM-^@M-^Y-extremity of a sequence encoding the 75 C-terminal residues of the viral VP16 transcriptional activator and inserted into the pG0229-35S:GR plant transformation vector between the CaMV-35S promoter and sequences encoding the rat glucocorticoid receptor (GR), so as to conserve the entire reading frame. The resulting plasmid was transferred to Agrobacterium tumefaciens C58/pMP90 cells and used to transform A. thaliana Col-0 plants by standard methods. A homozygous, single-copy transformant was identified, from which two populations of ten T3 descendents were grown and treated by dipping of inflorescences for 2 min in cycloheximide (CHX; 10 M-BM-5g/ml) containing Silwet L-77 surfactant (0.01% v/v). This treatment reduced translation to approximately 5% of its native level in inflorescence tissues, as measured by the in vivo incorporation of [35S]-methionine into proteins. One hour later, inflorescence tissues of the two plant populations were dipped for 2 min in CHX solution, as described above, with and without dexamethasone (DEX; 10 M-BM-5M). After a further 2h, treated inflorescences, excluding open flowers, were harvested and pooled prior to RNA extraction for global gene expression analyses. Microarray analyses were performed using CATMA microarrays each containing 31776 gene-specific tags corresponding to 22089 A. thaliana genes. Three biological replicates were performed, based on three entirely separate induction experiments involving T3 plants from the same T2 parent. A transformed line containing a construction in which SPT had been replaced by an initiation codon and nuclear localization signal (NLS) was used to verify that the targets identified did not respond to the nuclear translocation of VP16-GR protein alone (35S:NLS-VP16-GR plants). In this NLS control experiment, two biological replicates were performed. One technical replicate with fluorochrome reversal was performed for each biological replicate. The labeling of cRNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products), the hybridization of these cRNAs to microarrays, and the subsequent scanning of microarrays, was performed as described by Lurin et al. (2004) Plant Cell 16, 2089-2103.
Project description:For shade-intolerant plants, changes in light quality indicative of competition from neighboring plants trigger shade avoidance syndrome (SAS). PYHTOCHROME-INTERACTING FACTOR 7 is the major transcriptional regulator of SAS in Arabidopsis. However, the epigenetic reprogramming under shade is poorly understood. To identify the histone chaperone ASF1 and HIRA function during SAS, we performed transcriptome deep sequencing (RNA-seq) to search for differentially expressed genes (DEGs) by comparing transcript levels between Col-0 and pif7-1, asf1ab, or hira-1 seedlings during white light and shade conditions. Our data shown that histone chaperone ASF1, through interacting with PIF7 and helping of HIRA, positively regulates shade-induced genes expression.
Project description:ChIP-Seq were performed using 35S::GFP-ASF1A, pHTR13::HTR13-HA, pHTR5::HTR5-HA, and pHTR5::HTR5-HA/pif7-1 transgenic plants after white light and 1 hr shade treatment. Two biological replicates were prepared for each genotype of plants grown under light and shade conditions. High-confidence binding peaks with cut off fold change > 1.5 and P-value <0.01. Our data reveal that ASF1 directly regulating a subset of target genes of PIF7. Furthermore, shade-elicited gene activation is accompanied by H3.3 enrichment, which is mediated by the PIF7-ASF1 regulatory module.
Project description:In seedlings, the induction of shade avoidance syndrome (SAS) involves a rapid up-regulation for known shade marker genes and subsequently activates an interacting network of various hormones that will eventually lead to cell elongation. We found that the B-box protein AtBBX24 have positive effects on the SAS (positive regulators). Global expression analysis of col and bbx24 seedlings reveals that a large number of genes involved in hormonal signaling pathways are positively regulated by BBX24 in response to simulated shade. The microarray experiment consisted in a factorial design comprising two genotypes (col and bbx24-1) and two light treatments (white light and simulated shade). RNA material was extracted from 7-day-old seedlings. Two replicates per treatment were performed using Affymetrix Arabidopsis ATH1 GeneChips. RNA was prepared, labeled and hybridized in accordance with the manufacturerM-bM-^@M-^Ys instructions. Data were normalized and genes with M-bM-^@M-^XabsentM-bM-^@M-^Y calls and a signal of <50 units in all replicate experiments were filtered out. Significantly differentially expressed genes were identified by performing profile analysis using Significance Analysis of Microarrays (SAM; Tusher et al., 2001).
Project description:The goal of this study was to compare the transcriptional profile (RNA-seq) of Arabidopsis thaliana Columbia-0 ecotype seedlings that were treated with two different light (white light or shade) and temperature (20ºC or 28ºC) conditions.