Veränderungen im Transkriptom und Chromatin bei der Festlegung von Gründerzellen im Arabidopsis thaliana Infloreszenzmeristem

Die DRNL-Expression markiert Gründerzellen lateraler Seitenorgane im Infloreszenzmeristem (IM) von Arabidopsis thaliana. Für die Analyse des Transkriptoms sowie der Chroma¬tin¬struk¬tur der Grün¬derzellen wurden die DRNL-ex¬pri¬mie¬renden Zellen (GFP+) von den umge¬benden Zellen (GFP–) des apetala1-1 cauliflower 1 IMs getrennt. Sowohl die Genaktivität als auch die mittels „As¬say for Transpo¬sase-Accessible Chro¬¬ma¬tin with high¬trough¬put sequen¬¬cing” (ATAC-Seq) identifizierte Chromatinzugänglichkeit weisen genspezifische Unterschiede zwi¬schen den beiden Zelltypen auf. Gene mit Tn5 Transposase hypersensitive Stellen (THSs) weisen eine stärkere Transkription auf, dies spiegelt sich in der häufigen Lokalisation von THSs am Trans¬krip¬tions¬start wider. Für differenziell regulierte Gene konnte eine Korrelation mit einer erhöhten Chro¬ma¬tin¬zugäng¬lich¬keit entweder in GFP+ oder GFP– Zellen (dTHS) beobachtet werden. In GFP+ Zellen hochregulierte Gene weisen zugänglicheres Chromatin in den GFP+ Zellen (dTHS+) auf bzw. die herun¬ter¬re¬gu¬lier¬ten Gene in GFP– Zellen (dTHS–). Eine Zuord¬nung der Gene in Gen¬on¬tolo¬gie¬grup¬pen zeigt funktionelle Übereinstimmungen für die hoch¬regu¬lier¬ten und dTHS+ Genen bzw. die herun¬ter¬regu¬lier¬¬ten und dTHS– Genen. Die GFP+ Zellen haben Anteil an der Blüten¬ent¬wick¬lung und stehen damit im direk¬¬ten Zu¬sam¬men¬hang zu den DRNL-expri¬mie¬ren¬den Gründerzellen, die GFP– Zellen sind im Gegen¬satz zum großen Teil mit der Ant¬wort auf Stress assoziiert. Daher zeigen die Grün¬der¬¬zellen spezi¬fische Unterschiede in der Genaktivität in Korrelation mit der Chroma¬tin-kon¬for¬¬ma¬tion im Vergleich zu den restlichen Zellen des IMs. THSs zeigen eine hohe posi¬tio-nelle Über¬¬ein¬stimmung mit konservierten DNA-Sequenzen sowie AP1 und SEP3 Bindestellen; AP1 bindet eher dTHS+ Gene, SEP3 hin¬gegen dTHS– Gene. Die hohe Sensitivität der ATAC-Seq Metho¬de ermöglicht daher die Identi¬¬fikation von cis-regulatorischen Elementen, auch an Posi¬tionen die erst zu einem späteren Zeit¬punkt besetzt werden

Specific chromatin changes mark lateral organ founder cells in the Arabidopsis inflorescence meristem

Fluorescence-activated cell sorting (FACS) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were combined to analyse the chromatin state of lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis apetala1-1 cauliflower-1 double mutant inflorescence meristem. On a genome-wide level, we observed a striking correlation between transposase hypersensitive sites (THSs) detected by ATAC-seq and DNase I hypersensitive sites (DHSs). The mostly expanded DHSs were often substructured into several individual THSs, which correlated with phylogenetically conserved DNA sequences or enhancer elements. Comparing chromatin accessibility with available RNA-seq data, THS change configuration was reflected by gene activation or repression and chromatin regions acquired or lost transposase accessibility in direct correlation with gene expression levels in LOFCs. This was most pronounced immediately upstream of the transcription start, where genome-wide THSs were abundant in a complementary pattern to established H3K4me3 activation or H3K27me3 repression marks. At this resolution, the combined application of FACS/ATAC-seq is widely applicable to detect chromatin changes during cell-type specification and facilitates the detection of regulatory elements in plant promoters

Functional dissection of the DORNRoSCHEN-LIKE enhancer 2 during embryonic and phyllotactic patterning

Main conclusion The Arabidopsis DORNRoSCHEN-LIKE enhancer 2 comprises a high-occupancy target region in the IM periphery that integrates signals for the spiral phyllotactic pattern and cruciferous arrangement of sepals. Transcription of the DORNRoSCHEN-LIKE (DRNL) gene marks lateral organ founder cells (LOFCs) in the peripheral zone of the inflorescence meristem (IM) and enhancer 2 (En2) in the DRNL promoter upstream region essentially contributes to this phyllotactic transcription pattern. Further analysis focused on the phylogenetically highly conserved 100-bp En2(core) element, which was sufficient to promote the phyllotactic pattern, but was recalcitrant to further shortening. Here, we show that En2(core) functions independent of orientation and create a series of mutations to study consequences on the transcription pattern. Their analysis shows that, first, in addition to in the inflorescence apex, En2(core) acts in the embryo; second, cis-regulatory target sequences are distributed throughout the 100-bp element, although substantial differences exist in their function between embryo and IM. Third, putative core auxin response elements (AuxREs) spatially activate or restrict DRNL expression, and fourth, according to chromatin configuration data, En2(core) enhancer activity in LOFCs correlates with an open chromatin structure at the DRNL transcription start. In combination, mutational and chromatin analyses imply that En2(core) comprises a high-occupancy target (HOT) region for transcription factors, which implements phyllotactic information for the spiral LOFC pattern in the IM periphery and coordinates the cruciferous array of floral sepals. Our data disfavor a contribution of activating auxin response factors (ARFs) but do not exclude auxin as a morphogenetic signal

The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem

Background: Although the pattern of lateral organ formation from apical meristems establishes species-specific plant architecture, the positional information that confers cell fate to cells as they transit to the meristem flanks where they differentiate, remains largely unknown. We have combined fluorescence-activated cell sorting and RNA-seq to characterise the cell-type-specific transcriptome at the earliest developmental time-point of lateral organ formation using DORNROSCHEN-LIKE::GFP to mark founder-cell populations at the periphery of the inflorescence meristem (IM) in apetala1 cauliflower double mutants, which overproliferate IMs. Results: Within the lateral organ founder-cell population at the inflorescence meristem, floral primordium identity genes are upregulated and stem-cell identity markers are downregulated. Additional differentially expressed transcripts are involved in polarity generation and boundary formation, and in epigenetic and post-translational changes. However, only subtle transcriptional reprogramming within the global auxin network was observed. Conclusions: The transcriptional network of differentially expressed genes supports the hypothesis that lateral organ founder-cell specification involves the creation of polarity from the centre to the periphery of the IM and the establishment of a boundary from surrounding cells, consistent with bract initiation. However, contrary to the established paradigm that sites of auxin response maxima pre-pattern lateral organ initiation in the IM, auxin response might play a minor role in the earliest stages of lateral floral initiation

The AP2-type transcription factors DORNRA-SCHEN and DORNRA-SCHEN-LIKE promote G1/S transition

The paralogous genes DORNRA-SCHEN (DRN) and DORNRA-SCHEN-LIKE (DRNL) encode AP2-type transcription factors that are expressed and act cell-autonomously in the central stem-cell zone or lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis shoot meristem (SAM), but their molecular contribution is unknown. Here, we show using the Arabidopsis thaliana MERISTEM LAYER 1 promoter that DRN and DRNL share a common function in cell cycle progression and potentially provide local competence for G1-S transitions in the SAM. Analysis of double transgenic DRN::erGFP and DRNL::erCERULEAN promoter fusion lines suggests that the trajectory of this cellular competence starts with DRN activity in the central stem-cell zone and extends locally via DRNL activity into groups of founder cells at the IM or FM periphery. Our data support the scenario that after gene duplication, DRN and DRNL acquired different transcription domains within the shoot meristem, but retained protein function that affects cell cycle progression, either centrally in stem cells or peripherally in primordial founder cells, a finding that is of general relevance for meristem function

Additional file 1: of The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem

Comparison of log2-transformed expression from qRT-PCR for a subset of 18 up- or downregulated genes from the transcriptome dataset normalised to ACTIN2 expression and the log2 (relative expression) from RNA-seq. (DOC 150 kb

Additional file 2: of The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem

Primer sequences and list of genes used for real-time PCR expression analysis. (DOCX 147 kb