Project description:Cell-type specific transcriptional profiles were generated by FACS (Fluorescence Activated Cell Sorting) sorting of roots that express cell-type specific GFP-reporters. Five different GFP-reporter lines were utilized allowing us to obtain transcriptional profiles for cells in all radial zones of the root. FACS cell populations were isolated from roots grown under standard conditions or roots that had been transfered to -Fe media for 24 hours. Little is known about how developmental cues affect the way cells interpret their environment. Here we characterize the transcriptional response of different cell layers and developmental stages of the Arabidopsis root to high salinity and find that transcriptional responses are highly constrained by developmental parameters. These transcriptional changes lead to the differential regulation of specific biological functions in subsets of cell-layers, several of which correspond to observable physiological changes. We show that known stress pathways primarily control semi-ubiquitous responses and use mutants that disrupt epidermal patterning to reveal cell-layer specific and inter-cell-layer effects. By performing a similar analysis using iron-deprivation we identify common cell-type specific stress responses and environment-independent biological functions that define each cell type. Experiment Overall Design: To gain a genome-scale understanding of the role that developmental processes play in regulating stimulus response, we examined the effect of -Fe stress on gene expression along the radial axis of the root. Cell identity is the main variable that changes along the radial axis with the epidermis representing the outermost tissue layer and the stele representing the inner most layer. 5 different GFP reporter lines were used to isolate specific populations of cells from the Arabidopsis root using FACS sorting of protoplasted cells. GFP-reporter lines were exposed to iron deficient (-Fe) conditions (0.3mM Ferrozine in MS media containing no ferrous sulfate) for 24 hours before hand.

Project description:In order to estimate the effects of protoplasting and FACS sorting procedures on salt regulated gene expression we generated expression profiles for whole roots that had been treated with salt for 1 hour and for roots that were protoplasted and FACS sorted after the initial 1 hour salt treatment. Cells are amazingly adept at integrating both external and internal cues to regulate transcriptional states. While internal processes such as differentiation and cell-type specification are generally understood to have an important impact on gene expression, very little is known about how cells utilize these developmental cues to regulate responses to external stimuli. Here we use the response to a well characterized environmental stress, high salinity, to obtain a global view of the role that cell identity plays in guiding transcriptional responses in the root of Arabidopsis. Our analysis is based on three microarray data sets we have generated that explore transcriptional changes spatially among 6 cell layers and 4 longitudinal regions or temporally along 5 time points after salt treatment. We show that the majority of the response to salt stress is cell-type specific resulting in the differential regulation of unique biological functions in subsets of cell layers. To understand the regulatory mechanisms controlling these responses we have analyzed cis-element enrichment in the promoters of salt responsive genes and demonstrate that known stress regulatory elements likely control responses to salt occurring in multiple cell types. Despite the extensive shift in transcriptional state that salt stress elicits, we are able to identify several biological processes that consistently define each cell layer and find that transcriptional regulators of cell-identity tend to exhibit robust cell-type specific expression. Finally, using mutants that disrupt cell-type specification in the epidermis, we reveal cell autonomous and non-autonomous effects when cell identity is altered. Together, these data elucidate a novel intersection between physiology and development and expand our understanding of how transcriptional states are regulated in a multi-cellular context. Experiment Overall Design: To estimate the effect that protoplasting and cell sorting has on the expression of salt regulated genes we prepared samples as in Birnbaum et al. (2005) Nat. Methods, except that all cells were collected after cell sorting. Cells were collected from roots that had been treated with 140mM NaCl for 1hr prior to protoplasting. Whole roots were also collected after a similar treatment regimen with NaCl. Two replicates were performed per condition.

Project description:Cell-type specific transcriptional profiles were generated by FACS (Fluorescence Activated Cell Sorting) sorting of roots that express cell-type specific GFP-reporters. Six different GFP-reporter lines were utilized allowing us to obtain transcriptional profiles for cells in all radial zones of the root. FACS cell populations were isolated from roots grown under standard conditions or roots that had been transfered to media supplemented with 140 mM NaCl for 1 hour. Cells are amazingly adept at integrating both external and internal cues to regulate transcriptional states. While internal processes such as differentiation and cell-type specification are generally understood to have an important impact on gene expression, very little is known about how cells utilize these developmental cues to regulate responses to external stimuli. Here we use the response to a well characterized environmental stress, high salinity, to obtain a global view of the role that cell identity plays in guiding transcriptional responses in the root of Arabidopsis. Our analysis is based on three microarray data sets we have generated that explore transcriptional changes spatially among 6 cell layers and 4 longitudinal regions or temporally along 5 time points after salt treatment. We show that the majority of the response to salt stress is cell-type specific resulting in the differential regulation of unique biological functions in subsets of cell layers. To understand the regulatory mechanisms controlling these responses we have analyzed cis-element enrichment in the promoters of salt responsive genes and demonstrate that known stress regulatory elements likely control responses to salt occurring in multiple cell types. Despite the extensive shift in transcriptional state that salt stress elicits, we are able to identify several biological processes that consistently define each cell layer and find that transcriptional regulators of cell-identity tend to exhibit robust cell-type specific expression. Finally, using mutants that disrupt cell-type specification in the epidermis, we reveal cell autonomous and non-autonomous effects when cell identity is altered. Together, these data elucidate a novel intersection between physiology and development and expand our understanding of how transcriptional states are regulated in a multi-cellular context. Experiment Overall Design: To gain a genome-scale understanding of the role that developmental processes play in regulating stimulus response, we examined the effect of salt stress on gene expression along the radial axis of the root. Cell identity is the main variable that changes along the radial axis with the epidermis representing the outermost tissue layer and the stele representing the inner most layer. 6 different GFP reporter lines were used to isolate specific populations of cells from the Arabidopsis root using FACS sorting of protoplasted cells. GFP-reporter lines were grown under standard conditions before protoplasting and sorting or they were transfered to media supplemented with 140mM NaCl for 1 hour before hand.

Project description:To gain a genome-scale understanding of the role that developmental processes play in regulating stimulus response, we examined the effect of -Fe stress on gene expression along the longitudinal axis of the root. Since roots grow from stem cells located near the tip, the position of cells along the longitudinal axis can be used as a proxy for developmental time, with distance from the root tip correlating with increased differentiation. To estimate the role developmental stage plays in regulating salt response, roots were dissected into four longitudinal zones (LZ data set) after transfer to standard or -Fe media and transcriptionally profiled. Little is known about how developmental cues affect the way cells interpret their environment. Here we characterize the transcriptional response of different cell layers and developmental stages of the Arabidopsis root to high salinity and find that transcriptional responses are highly constrained by developmental parameters. These transcriptional changes lead to the differential regulation of specific biological functions in subsets of cell-layers, several of which correspond to observable physiological changes. We show that known stress pathways primarily control semi-ubiquitous responses and use mutants that disrupt epidermal patterning to reveal cell-layer specific and inter-cell-layer effects. By performing a similar analysis using iron-deprivation we identify common cell-type specific stress responses and environment-independent biological functions that define each cell type. Experiment Overall Design: Roots were grown under standard conditions for 5 days then transfered to standard media or iron deficient (-Fe) conditions (0.3mM Ferrozine in MS media containing no ferrous sulfate). 24 hours after transferring seedlings, roots were cut into 4 regions using a razor blade. The first cut was made ~150 Âµm from the root tip at the point where the shape of the root transitions from conical to cylindrical (Zone 1). The second cut was made ~200 Âµm above the first cut, at the point were the root becomes less optically dense, which marks the approximate end of the meristematic zone (Zone 2). The third cut was made ~200-300 Âµm above the second cut, just below the region where root hairs begin to emerge (Zone 3). The fourth cut was made ~1 mm above the third cut (Zone 4).

Dataset Information

Expression analysis of the effect of protoplasting and FACS sorting in roots exposed to iron deficiency (-Fe)

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