Journal: Development (Cambridge, England)

Article Title: Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network

doi: 10.1242/dev.157081

Figure Lengend Snippet: Transcriptomic analysis of timecourse data. (A) STM expression (mRNA levels following induction with DEX. For each time-point, the STMoe line (S3.8) was compared with an empty-vector control line (23.7) under identical induction conditions. Purple bars indicate STM mRNA levels, as measured on an Affymetrix ATH1 microarray using RMA/LIMMA with P <0.01. Blue bars indicate STM mRNA levels, as measured by QRT-PCR. Error bars indicate s.d. Three biological replicates were used for each time-point. (B) Morphology of STMoe timecourse seedlings and comparison of DEGs to identify exclusive overlaps at each time-point. A P -value cut-off of <0.01 was used to identify DEGs. Total number of DEGs is shown below each time-point. (C) (Top) DEG overlaps at each time-point using P <0.01 and a minimum 2-fold expression change filter. (Bottom) Comparison of DEGs to identify overlaps at successive time-points. Numbers in bold represent total number of DEGs with P <0.01 and a minimum 2-fold expression change at the respective time-point. Genes showing reciprocal expression dynamics between time-points were omitted. (D,E) Gene ontology (GO) SLIM enrichment analysis of DEGs identified at each STMoe time-point. All nodes were set to the same size for clarity. Progressive enrichment of GO categories is evident across the timecourse for biological process (D) and molecular function (E). No significant enrichment was found for molecular function at the STMoe 8 h time-point. Enriched GO categories are circled and given a generalised annotation term for simplicity. The full list of enriched GO categories is shown in Fig. S1 .

Article Snippet: Arrows indicate that STM acts as a parent node to CUC1, AIL7 and CHR40. (E) Quantification of mRNA levels of key network genes following 3 h induction of STM-GR fusion protein with 60 µm DEX compared with mock-induced sample (black bars) or DEX+CHX compared with CHX-treated sample (grey bars) using Affymetrix ATH1 DNA microarrays.

Techniques: Expressing, Plasmid Preparation, Microarray, Quantitative RT-PCR

Journal: Development (Cambridge, England)

Article Title: Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network

doi: 10.1242/dev.157081

Figure Lengend Snippet: STM Bayesian network analysis. (A) Consensus Bayesian network analysis of STM-responsive TFs identified by meta-analysis. Nodes in red indicate genes showing upregulation in response to increased STM activity, blue nodes indicate downregulation. Grey nodes indicate genes with variable differential expression across the timecourse. Darker lines (edges) indicate frequency of prediction: 40% (light grey) to 100% (black). Edge significance is the sum of both possible directions. Green outer circles indicate potential direct STM target as based on microarray data (see Materials and Methods). Fifty-four out of 57 transcription-associated genes show inter-connections (>40%) with the rest of the network. (B) Consensus Bayesian network analysis showing earliest response times in STMoe timecourse. Red, STMoe 8 h; dark orange, STMoe 24 h; light orange, STMoe 72 h; yellow, STMoe 9 days; blue, STM-RNAi 72 h or 9 days. (C) Expression patterns based on re-analysis of the data from . Green, expressed specifically in SAM; purple, expressed specifically in organ primordia; blue, expressed in SAM and organ primordia, either pWUS and pFIL domains (corpus) or pCLV3 and pFIL domains (tunica); grey, general non tissue-specific expression. (D) Local network of STM and the three most high-confidence associations. Arrows indicate that STM acts as a parent node to CUC1, AIL7 and CHR40. (E) Quantification of mRNA levels of key network genes following 3 h induction of STM-GR fusion protein with 60 µm DEX compared with mock-induced sample (black bars) or DEX+CHX compared with CHX-treated sample (grey bars) using Affymetrix ATH1 DNA microarrays. Three biological replicates were performed. Asterisks indicate significant ( P <0.01) and >2-fold expression changes. (F) Quantification of mRNA levels as in E using qRT-PCR. Three biological replicates were performed. (G) Hierarchical clustering (average distance UPGMA) of 57 meta-analysis DEGs encoding mostly transcriptional regulators in STMoe and STM-RNAi timecourse. Red indicates an increase in gene expression, blue indicates a decrease in gene expression. (H) ChIP analysis of STM-GR binding to gene promoter sequences. Gene promoters are shown schematically, with amplicons shown as grey boxes. IP samples were compared with mock-IP samples. Relative enrichment of fragments by qRT-PCR is shown on overlying graphs compared with control ACTIN2 amplicon. At least three biological replicates were performed for CUC1 and AIL7 . Two biological replicates were performed for AtHB25 and BOP2. All error bars indicate s.d.

Article Snippet: Arrows indicate that STM acts as a parent node to CUC1, AIL7 and CHR40. (E) Quantification of mRNA levels of key network genes following 3 h induction of STM-GR fusion protein with 60 µm DEX compared with mock-induced sample (black bars) or DEX+CHX compared with CHX-treated sample (grey bars) using Affymetrix ATH1 DNA microarrays.

Techniques: Activity Assay, Expressing, Microarray, Quantitative RT-PCR, Binding Assay, Amplification

Journal: Development (Cambridge, England)

Article Title: Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network

doi: 10.1242/dev.157081

Figure Lengend Snippet: Regulatory interactions among STM, CUC1, TCPs and miR164c. (A) qRT-PCR timecourse analysis of STM and CUC1 expression levels in DEX-induced CUC1-GR and STM-GR lines relative to mock-induced control lines. (B) qRT-PCR analysis of STM and CUC1 expression levels in long-term induced STM-GR and CUC1-GR lines relative to wild-type control lines. (C) qRT-PCR analysis of STM expression levels in response to induction of CUC1-GR with DEX versus mock induction or with cycloheximide (CHX) and DEX versus CHX. CHX-DEX treatment was discontinued after 16 h owing to toxicity effects. (D) pSTM:GUS (top) and pCUC1:GUS (bottom) activity (blue) in wild-type and STMoe or CUC1-GR (CUC1oe) seedlings treated with DEX. Arrows indicate GUS activity. (E) ChIP analysis of the upstream region (promoter) of STM . (Top) Schematic representation of positions of tested amplicons upstream of START codon. (Bottom) Quantification of ChIP amplicons in the IP sample relative to mock-IP sample. Results are based on two biological replicates. (F) qRT-PCR analysis of STM, CUC1 and pri-miR164c expression in STMoe samples 24 h after induction with DEX (STMoe 24 h DEX) and in plants treated with DEX from germination (STMoe constitutive DEX). (G) Expression of pmiR164c:GUS (top) and pmiR164c:VENUS (bottom) in wild-type and STMoe seedlings (arrow=SAM) treated with DEX. A pmiR164c:VENUS reporter is expressed ectopically in cotyledon bases in STMoe. Arrows indicate GUS or GFP activity. (H,I) Expression levels of STM , CUC1 , TCP3 and TCP4 in STMoe timecourse measured by ATH1 DNA microarray (H) and by qRT-PCR (I) in separate experiments. All experiments are based on at least three biological replicates unless otherwise stated. Error bars indicate s.d. All expression changes in H are significant ( P <0.01) except for TCP3 and TCP4 in the STMoe 8 h sample.

Article Snippet: Arrows indicate that STM acts as a parent node to CUC1, AIL7 and CHR40. (E) Quantification of mRNA levels of key network genes following 3 h induction of STM-GR fusion protein with 60 µm DEX compared with mock-induced sample (black bars) or DEX+CHX compared with CHX-treated sample (grey bars) using Affymetrix ATH1 DNA microarrays.

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Microarray

Journal: PLoS Biology

Article Title: Epigenomic Consequences of Immortalized Plant Cell Suspension Culture

doi: 10.1371/journal.pbio.0060302

Figure Lengend Snippet: A Venn diagram of transcripts with significant TE homology that show significant overaccumulation (normalized value ≥ 1.5) in ATH1-based microarray expression profiles of cell suspension culture, seedling, and callus samples. There is no overlap between samples and a much higher degree of TE activity in cell suspension culture.

Article Snippet: Transcriptional reactivation of TEs was analyzed by microarray analysis using Affymetrix Arabidopsis ATH1 microarrays.

Techniques: Microarray, Expressing, Activity Assay

Journal: PLoS Biology

Article Title: Epigenomic Consequences of Immortalized Plant Cell Suspension Culture

doi: 10.1371/journal.pbio.0060302

Figure Lengend Snippet: The relative frequencies of all sRNAs (with all known miRNA sequences removed) according to their size for each of the small RNA libraries, as well as only for those sRNAs matching particular families of Arabidopsis repeats ( AR12 , Athila , Atlantys , Helitron , AtGP1 , AtMu , AtENSPM , AtCopia , and AtVandal ).

Article Snippet: Transcriptional reactivation of TEs was analyzed by microarray analysis using Affymetrix Arabidopsis ATH1 microarrays.

Techniques:

Journal: PLoS Biology

Article Title: Epigenomic Consequences of Immortalized Plant Cell Suspension Culture

doi: 10.1371/journal.pbio.0060302

Figure Lengend Snippet: Significant gene expression differences detected by ATH1 microarray expression profiling between cell suspension culture and seedling or callus samples are represented in this Venn diagram. Also, 65% of genes annotated by ChromDB as having a role in chromatin regulation are differentially expressed in these samples relative to seedling.

Article Snippet: Transcriptional reactivation of TEs was analyzed by microarray analysis using Affymetrix Arabidopsis ATH1 microarrays.

Techniques: Expressing, Microarray