Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Arabidopsis mesophyll cells to identify KIN10-target genes

ABSTRACT: The goal of this experiment was to explore the extent of KIN10 (At3g01090) transcriptional regulation and identify its early target genes in Arabidopsis mesophyll protoplasts. Results suggest that KIN10 targets a remarkably broad array of genes that orchestrate transcription networks, promote catabolism and autophagy, and suppress anabolism and ribosome biogenesis. The transient expression condition ruled out secondary or long-term effects of metabolism and growth, and circumvented experimental limitations caused by redundancy and embryonic lethality observed in mammals and plants. Experiment Overall Design: Analysis of the transcript changes induced by transient KIN10 (At3g01090) expression (6h) in Arabidopsis mesophyll protoplasts using protoplasts similarly transfected with control plasmid DNA for comparison. To maximize the significance of the duplicated experimental data for defining differentially expressed genes, the biologically independent experiments were carefully performed by two individuals using different soil, plants, growth chambers, DNAs and microarray facilities for hybridization and scanning to cover potential technical and biological variations. For each pair (control & KIN10) of large-scale protoplast transfection experiments, about 3 million protoplasts were isolated from the fifth and sixth leaves of 36 randomly picked plants and pooled. Experiment Overall Design: RNA samples isolated from control and KIN10-transfected protoplasts were subjected to quality controls, including RNA quality and quantity inspection by gel electrophoresis and staining as well as consistent gene expression changes by duplicated real-time quantitative RT-PCR analyses of selected marker genes.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Elena Baena-Gonzalez

PROVIDER: E-GEOD-8257 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A central integrator of transcription networks in plant stress and energy signalling.

Baena-González Elena E Rolland Filip F Thevelein Johan M JM Sheen Jen J

Nature 20070801 7156

Photosynthetic plants are the principal solar energy converter sustaining life on Earth. Despite its fundamental importance, little is known about how plants sense and adapt to darkness in the daily light-dark cycle, or how they adapt to unpredictable environmental stresses that compromise photosynthesis and respiration and deplete energy supplies. Current models emphasize diverse stress perception and signalling mechanisms. Using a combination of cellular and systems screens, we show here that ...[more]

PMID: 17671505

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Project description:BACKGROUND:Dynamic transcriptional regulation is critical for an organism's response to environmental signals and yet remains elusive to capture. Such transcriptional regulation is mediated by master transcription factors (TF) that control large gene regulatory networks. Recently, we described a dynamic mode of TF regulation named "hit-and-run". This model proposes that master TF can interact transiently with a set of targets, but the transcription of these transient targets continues after the TF dissociation from the target promoter. However, experimental evidence validating active transcription of the transient TF-targets is still lacking. RESULTS:Here, we show that active transcription continues after transient TF-target interactions by tracking de novo synthesis of RNAs made in response to TF nuclear import. To do this, we introduced an affinity-labeled 4-thiouracil (4tU) nucleobase to specifically isolate newly synthesized transcripts following conditional TF nuclear import. Thus, we extended the TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors) to include 4tU-labeling and named this new technology TARGET-tU. Our proof-of-principle example is the master TF Basic Leucine Zipper 1 (bZIP1), a central integrator of metabolic signaling in plants. Using TARGET-tU, we captured newly synthesized mRNAs made in response to bZIP1 nuclear import at a time when bZIP1 is no longer detectably bound to its target. Thus, the analysis of de novo transcripomics demonstrates that bZIP1 may act as a catalyst TF to initiate a transcriptional complex ("hit"), after which active transcription by RNA polymerase continues without the TF being bound to the gene promoter ("run"). CONCLUSION:Our findings provide experimental proof for active transcription of transient TF-targets supporting a "hit-and-run" mode of action. This dynamic regulatory model allows a master TF to catalytically propagate rapid and broad transcriptional responses to changes in environment. Thus, the functional read-out of de novo transcripts produced by transient TF-target interactions allowed us to capture new models for genome-wide transcriptional control. Root protoplasts were transfected with 35S::GR::bZIP1 or Empty vector (EV=pJD385_35S::GR) constructs and cells were sequentially treated with: i) the nitrogen signal (N), ii) cycloheximide (CHX), iii) dexamethasone (DEX; for review see Bargmann et. al 2013). Cells were exposed to 4tU to affinity-label newly synthesized transcripts prior RNA extraction followed by specific pull down of 4tu-fractions and hybridization on Arabidopsis ATH1 microarray chips.

Project description:The organs of multicellular species are comprised of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change but little is known about how responses are tailored to specific cell types or coordinated among them on a global level. Here we use cellular profiling of five Arabidopsis root cell types in response to a limiting resource, nitrogen, to uncover a vast and predominantly cell-specific response that was largely undetectable using traditional methods. These methods reveal a new class of cell-specific nitrogen responses. As a proof-of-principle, we dissected one cell-specific response circuit that mediates nitrogen-induced changes in root branching from pericycle cells. Thus, cellular response profiling links gene modules to discrete functions in specific cell types. Experiment Overall Design: The whole experiment was carried out in triplicate with 84 chips in total (28 experiments). The nitrogen response in 5 FACS-sorted root cell types (LRC, lateral root cap; EpiC, epidermis and cortex; EndoP, endodermis and pericycle; Peri, pericycle; Stele, stele), protoplasts and Col-0 whole roots was assayed using microarrays. 5mM KNO3 was used as a nitrogen treatment for 2 hours. 5mM KCl was used as a control treatment for the same length of time. 1mM MSX in combination with 5mM KNO3 (or with 5mM KCl as a control) was used to block the assimilation of KNO3, and 5mM Gln added to restore it in order to separate KNO3 effects from assimilated-nitrogen effects. 5mM KNO3 was either maintained in the protoplast-generating solution (continuous treat) or not (transitory treat) to test how to best preserve the cellular nitrogen-response during the 1 hr protoplast generating procedure. As controls we used protoplasts, whole roots frozen directly after the 2hr treatment or 3.5hr treatment, or whole roots incubated in protoplast-generating solution (minus enzymes; transitory or continuous treated) and then frozen. After FACS-sorting or mock FACS-sorting cells were frozen.

Dataset Information

Transcription profiling of Arabidopsis mesophyll cells to identify KIN10-target genes

Publications

A central integrator of transcription networks in plant stress and energy signalling.

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