Project description:This experiment was set up in order to identify the (direct) transcriptional targets of the Ethylene Response Factor 115 (ERF115) transcription factor. Because ERF115 expression occurs in quiescent center (QC) cells and strong effects on the QC cells were observed in ERF115 overexpression plants, root tips were harvested for transcript profiling in order to focus on root meristem and QC specific transcriptional targets.
Project description:The Arabidopsis quiescent center (QC) is a small group of cells with low mitotic activity located at the center of the root stem cell niche. Its transcriptional profile was previously analyzed using two repeats of cells FACS isolated using the WOX5 marker. To get more power in analyzing QC transcriptional profile, we generated three additional samples of the QC, using the QC-specific marker WOX5.
Project description:This experiment was set up in order to identify the (direct) transcriptional targets of the Ethylene Response Factor 115 (ERF115) transcription factor. Because ERF115 expression occurs in quiescent center (QC) cells and strong effects on the QC cells were observed in ERF115 overexpression plants, root tips were harvested for transcript profiling in order to focus on root meristem and QC specific transcriptional targets. Wild-type (Col-0 ecotype), erf115 mutant (SALK_021981) and ERF115 overexpressing (p35S:ERF115 ORF) root tips (three replicates each) were harvested and subjected to transcript profiling, using the Col-0 samples as control reference.
Project description:The Arabidopsis quiescent center (QC) is a small group of cells with low mitotic activity located at the center of the root stem cell niche. Its transcriptional profile was previously analyzed using two repeats of cells FACS isolated using the AGL42 marker. To get more power in analyzing QC transcriptional profile, we generated three additional samples of the QC, using the QC-specific marker WOX5. Three replicates of FACS-sorted GFP-positive cells from WOX5:GFP roots.
Project description:In multicellular organisms, communication between cells is vital for their fate determination. In plants, the quiescent center (QC) signals to adjacent stem cells to maintain them undifferentiated. However, how surrounding stem cells instruct the QC remains poorly understood. Here we show that in the Arabidopsis root, microRNA160 (miR160) moves from vascular stem cells to the QC, where it degrades the mRNAs of two auxin response factors, ARF10 and ARF17. This degradation relieves BRAVO from direct transcriptional repression, maintaining QC quiescence. We further identify that blocking miR160 movement due to DNA damage-induced vascular stem cell death and restricted symplastic transport reduces BRAVO and WOX5 expression, leading to QC division to replenish damaged stem cells during root regeneration. Together, our results demonstrate that a transcriptional axis initiated by mobile miR160 regulates the QC and stem cell behavior, advancing our understanding of the communication between stem cells and their surrounding cellular environment.
Project description:We report the discovery of a root growth program in Arabidopsis that is independent of a functional quiescent center (QC). In this regulatory program, PHABULOSA (PHB), posttranscriptionally regulated by SHR and SCR, plays a central role. In phb shr and phb scr mutants, root meristem/growth activity recovers significantly. Interestingly, this recovery does not accompany the resurgence of QC cells. PHB regulates apical root growth in stele cells of the root meristem, located proximal to the QC. Our genome-wide investigation suggests that PHB exerts its influence on root growth by regulating auxin-cytokinin homeostasis. Apical root growth was restored when cytokinin levels were genetically reduced in the shr mutant. Conversely, when miRNA-resistant PHB was expressed in the root stele cells, apical root growth and meristem functions were significantly inhibited without blocking the QC identity. Taken together, our investigation reveals two mechanisms through which SHR regulates root growth and stem cell activities: one is to specify and maintain the QC and the other is to regulate the proximal meristem activity through PHB and cytokinin. In this regulation, QC seems to be more involved in maintaining the “growth signal” and thus ensure the indeterminate root growth.
Project description:To demonstrate our method of controlling for technical noise in single-cell RNA-seq experiments we manually collected single A. thaliana cells marked by the expression of green fluorescent protein (GFP) driven by either the GL2 or WOX5 promoters. Seven and six cells were collected from each cell type, respectively. The GL2 promoter marks the non-hair cells in the root epidermis whereas the WOX5 promoter specifies the quiescent center (QC) of the root. Each cell selected was processed together with 50 pg of total HeLa RNA spike-in to prepare RNA-seq libraries using the Tang protocol. For comparison, we again added the commercially available ERCC spike-ins. We also performed RNA-seq on a set of technical replicates of total A. thaliana RNA using starting amounts ranging from 5000pg down to 10pg, a range that covers the RNA content obtainable from single cells of various sizes.
Project description:The quiescent center (QC) plays an essential role during root development by creating a microenvironment that preserves the stem cell fate of its surrounding cells. Strikingly, in order to retain root structure, QC cells only occasionally self-renew, displaying a proliferation rate far below that of all other cells within the root meristem. Previously, the APC/CCCS52A2 ubiquitine ligase and brassinosteroid signaling pathways have been found to antagonistically control Arabidopsis thaliana QC cell proliferation. Here, we demonstrate that both pathways converge on the ERF115 transcription factor that acts as a rate-limiting factor of QC cell division through transcriptional control of the autocrine phytosulfokine PSK5 peptide hormone. ERF115 marks QC cell division but is restrained through proteolysis by the APC/CCCS52A2 ubiquitine ligase, whereas QC proliferation is driven by brassinosteroid-dependent ERF115 expression. Combined, these two antagonistic mechanisms delimit the ERF115-PSK5 activity and QC renewal. Our results reveal a unique cell cycle regulatory mechanism that accounts for the low proliferation rate of QC cells within a surrounding population of highly mitotic active cells.