Project description:Asymmetric division of cortex/endodermal initials (CEI) in the Arabidopsis root generates two layers of ground tissue and is controlled by a finely orchestrated interplay between the transcription factors, SHORT ROOT (SHR) and SCARECROW (SCR). To understand the dynamics of the SHR/SCR regulatory network we performed microarray time course experiments using inducible versions of SHR and SCR and examined their transcriptional effects specifically in the ground tissue. Keywords: SHR and SCR time course and CEI cell-type analyses
Project description:Asymmetric division of cortex/endodermal initials (CEI) in the Arabidopsis root generates two layers of ground tissue and is controlled by a finely orchestrated interplay between the transcription factors, SHORT ROOT (SHR) and SCARECROW (SCR). To understand the dynamics of the SHR/SCR regulatory network we performed microarray time course experiments using inducible versions of SHR and SCR and examined their transcriptional effects specifically in the ground tissue. Keywords: SHR and SCR time course and CEI cell-type analyses We performed a time course analysis (TC data set) of the ground tissue (sorted J0571 enancher trap) after transfer either SHR or SCR inducible plants to Dex containing plates 0, 1, 3, 6 and 12 hours. We sorted for CEI cells using the pCYCD6::GFP marker line. Seedlings were grown for 5 days. SHR and SCR inducible plants were transfer to 1microM Dex MS plates. J0571 and CYCD6 sorted fluorecent cells were harvested for RNA extraction.
Project description:SHORT-ROOT (SHR) and SCARECROW (SCR) are required for stem cell maintenance in the Arabidopsis thaliana root meristem, ensuring its indeterminate growth. Mutation of SHR and SCR genes results in disorganization of the quiescent center and loss of stem cell activity, resulting in the cessation of root growth. This manuscript reports on the role of SHR and SCR in the development of leaves, which, in contrast to the root, have a determinate growth pattern and lack a persistent stem-cell niche. Our results demonstrate that inhibition of leaf growth in shr and scr mutants is not a secondary effect of the compromised root development, but is caused by a direct effect on cell division in the leaves: a reduced cell division rate and early exit of proliferation phase. Consistent with the observed cell division phenotype, the expression of SHR and SCR genes in leaves is closely associated with cell division activity in most cell types. The increased cell cycle duration is due to a prolonged S-phase duration, which is mediated by up-regulation of cell cycle inhibitors known to restrain the activity of the transcription factor, E2Fa. Therefore, we conclude that, in contrast to their specific role in cortex/endodermis differentiation and stem cell maintenance in the root, SHR and SCR primarily function as general regulators of cell proliferation in leaves.
Project description:SHORT-ROOT (SHR) and SCARECROW (SCR) are required for stem cell maintenance in the Arabidopsis thaliana root meristem, ensuring its indeterminate growth. Mutation of SHR and SCR genes results in disorganization of the quiescent center and loss of stem cell activity, resulting in the cessation of root growth. This manuscript reports on the role of SHR and SCR in the development of leaves, which, in contrast to the root, have a determinate growth pattern and lack a persistent stem-cell niche. Our results demonstrate that inhibition of leaf growth in shr and scr mutants is not a secondary effect of the compromised root development, but is caused by a direct effect on cell division in the leaves: a reduced cell division rate and early exit of proliferation phase. Consistent with the observed cell division phenotype, the expression of SHR and SCR genes in leaves is closely associated with cell division activity in most cell types. The increased cell cycle duration is due to a prolonged S-phase duration, which is mediated by up-regulation of cell cycle inhibitors known to restrain the activity of the transcription factor, E2Fa. Therefore, we conclude that, in contrast to their specific role in cortex/endodermis differentiation and stem cell maintenance in the root, SHR and SCR primarily function as general regulators of cell proliferation in leaves. Total RNAs were extracted using RNeasy plant mini kit (Qiagen) from vegetative part of seedlings at stage 1.04 grown in vitro. RNAs from three biological repeats of wild type (wt, control) and scr and shr mutants were submitted to ATH1 array hybridization.
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:Transcriptional profiling of the vegetative part of Arabidopsis comparing wild type with the shr scl23 scr triple mutant. The latter is produced by crossing the strong null alleles of shr (shr-2), scl23 (scl23-1) and scr (scr-5). The goal was to determine the effects of the GRAS transcription factors SHR, SCL23 and SCR on growth and development of the Arabidopsis shoot system by global transcriptome analysis.
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 M-bM-^@M-^\growth signalM-bM-^@M-^] and thus ensure the indeterminate root growth. Total 7 samples (2 replicates of shr-2 mutant (high PHABULOSA expression) vs. 2 replicates of shr-2 phb-6 (low/absent PHABULOSA expression). 3 replicates of Wild type used as reference sample.
Project description:Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning during multicellular organ development. In the Arabidopsis root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells and the other for proto- and metaphloem sieve elements. The molecular mechanisms underlying each of these processes have been reported, however, how these two are coordinated has remained elusive. Here, we report that SHORTROOT (SHR) is the key coordinator of two ACD processes for phloem development. SHR moving into the endodermis regulates ACD for companion cells by turning on microRNA165/6. ACD that generates phloem sieve elements is mediated by SHR moving into phloem precursors. To find the downstream targets of SHR in the stele, pCRE1::erGFP was introduced into wild type and shr-2 backgrounds to express GFP in the stele cells in the root meristem. We then collected GFP-expressing stele cells from the wild type, shr-2 and pCRE1::SHRΔNLELDV:nlsGFP; shr-2 through Fluorescence Activated Cell Sorter (FACS). RNAs were extracted from the sorted cells of each line and processed for generating labeled probes to be hybridized onto GeneChip Arabidopsis Tiling 1.0R Array (Affymetrix). We then examined the influence of SHR on phloem enriched genes in the tiling array data. In this analysis, we found 224 genes that are down-regulated in shr-2 in comparison to the wild type and then restore expression in pCRE1::SHRΔNLELDV:nlsGFP; shr-2.
Project description:Transcriptional profiling of the vegetative part of Arabidopsis comparing wild type with the shr scl23 scr triple mutant. The latter is produced by crossing the strong null alleles of shr (shr-2), scl23 (scl23-1) and scr (scr-5). The goal was to determine the effects of the GRAS transcription factors SHR, SCL23 and SCR on growth and development of the Arabidopsis shoot system by global transcriptome analysis. Two-condition experiment: Col-0 vs. shr scl23 scr triple mutant. Biological replicates: 2 WT vs. triple mutant replicates, 2 WT vs. triple mutant replicates dye-swap replicates.
Project description:SHR is a key regulator of stem cell renewal and radial patterning in the Arabidopsis root. In previous studies we showed that the SHR is a transcriptional regulator and it regulates gene transcription directly. To fully understand the SHR developmental pathway, we aim to identify its direct target at the genome scale using the ChIP-on-chip method. To this end, we have designed a promoter microarray for Arabidopsis, which contains probes that tile the intergenic regions as well as the first intron and the 3' UTR for all annotated genes including miRNA genes. Chromatin immunoprecipitation (ChIP) was performed using an anti-GFP antibody on the root of a transgenic line expressing under the SHR promoter a functional fusion protein between SHR and GFP. After labeling with Cy5 and Cy3 respectively, DNA recoverd from the ChIP and mock experiments was hybridized to the same microarray. The array was scanned using an Agilent scanner and the signal intensity for each channel was retrieved and normalized by the Agilent feature extraction software.