Project description:This SuperSeries is composed of the following subset Series:; GSE10496: Expression analysis of the effect of protoplasting and FACS sorting in roots exposed to iron deficiency (-Fe); GSE10497: Expression analysis of root developmental zones after iron deficiency (-Fe) treatment; GSE10501: Expression analysis of root cell-types after iron deficiency (-Fe) treatment; GSE10502: Time course expression analysis of the iron deficiency (-Fe) response in Arabidopsis roots Experiment Overall Design: Refer to individual Series
Project description:In order to estimate the effects of protoplasting and FACS sorting procedures on -Fe regulated gene expression we generated expression profiles for whole roots that had been treated with -Fe for 24 hours and for roots that were protoplasted and FACS sorted after the initial 24 hour -Fe treatment. 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 estimate the effect that protoplasting and cell sorting has on the expression of -Fe 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 exposed to iron deficient (-Fe) conditions (0.3mM Ferrozine in MS media containing no ferrous sulfate) for 24 hours prior to protoplasting. Whole roots were also collected after a similar treatment regimen with -Fe. Three replicates were performed per condition.
Project description:In order to estimate the effects of protoplasting and FACS sorting procedures on -Fe regulated gene expression we generated expression profiles for whole roots that had been treated with -Fe for 24 hours and for roots that were protoplasted and FACS sorted after the initial 24 hour -Fe treatment. 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. Keywords: effects of protoplasting and FACS sorting
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:We performed small RNA-seq (sRNA-seq) study of Arabidopsis shoots under iron-sufficient (+Fe), iron deficient (-Fe) and iron resupply (Fe resupply) conditions to investigate and identify sRNAs whose expression is regulated by iron deficiency.
Project description:Arabidopsis wild-type plants (Col-0 accession) were grown on control (+Fe+P) for 7 days on 0.1X MS then transferred to three different medium: control (+Fe+P), iron deficiency (-Fe+P), and iron and phosphate deficiency conditions (-Fe-P). Shoots were collected 39 h, 52 h and 76 h after the transfer. For RNA-seq experiments, three biological replicates were used for each time point (39h, 52h and 76h) and each condition (+Fe+P, -Fe+P and -Fe-P) for a total of 27 samples.
Project description:Essential metals such as iron are required for healthy plant growth. Fe is an important cofactor and catalytic element in many biological processes. Fe and other metals can also be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalisation. The NAS genes are supposed to play an important role in Fe homeostasis. They are coding for enzymes called nicotianaminesynthase (NAS), which synthesize nicotianamine (NA) by a one-step condensation reaction of three molecules S-adenosyl-methionine. NA acts as a chelator for Fe, Cu, Ni and Zn and might be involved in the transport and allocation of Fe throughout the plant. We generated quadruple T-DNA insertion mutant nas plants to investigate NA function as described in Klatte et al., 2009, Plant Physiol. The nas4x-1 plants show an interveinal leaf chlorosis when turning from vegetative to reproductive stage, which intensifies when growing under Fe deficiency conditions. nas4x-1 plants have strongly reduced NA contents and show an elevated Fe deficiency response in roots. By performing microarray experiments we want to reveal global changes on transcriptional level in roots and leaves of nas4x-1 mutant compared to wild type plants grown under Fe supply and Fe deficiency conditions, respectively. The loss of NAS genes has a strong impact on the regulation of other metal homeostasis genes and allows to draw conclusions about nicotianamine function in metal homeostasis of A.thaliana. For this study, four-week old nas4x-1 mutant and wild type plants were exposed for 7 days to plant medium with and without Fe supply. These conditions have been established previously and have resulted in a reproducibly strong interveinal leaf chlorosis of nas4x-1 plants compared to wild type, especially upon Fe deficiency conditions. The experiment was repeated three times in consecutive weeks to obtain three independent biological repetitions. Rosette leaves and roots of five week-old plants were harvested, RNA was isolated and microarray hybridization was performed. 24 Total samples were analyzed. We generated the following pairwise comparisons: WT + Fe vs. – Fe, nas4x-1 + Fe vs. – Fe, + Fe WT vs. + Fe nas4x-1, - Fe WT vs. - Fe nas4x-1, roots and leaves