Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data from Arabidopsis thaliana

ABSTRACT: Nitrogen and light are two major regulators of plant metabolism and development. While genes involved in the control of each of these signals have begun to be identified, regulators that integrate gene responses to nitrogen and light signals have yet to be determined. Here, we evaluate the role of bZIP1, a transcription factor involved in light and nitrogen sensing, by exposing wild-type (WT) and bZIP1 T-DNA null mutant plants to a combinatorial space of N and L treatment conditions. We use ANOVA analysis combined with clustering and Boolean modeling, to evaluate the role of bZIP1 in mediating L and N signaling genome-wide. Arabidopsis thaliana were growth on basal MS salts (custom-made; GIBCO) with 0.5 mM KNO3, 3 mM sucrose and 0.8% BactoAgar at pH 5.7. After 14 days under long-day (16 h light: 8 h dark) conditions with light intensity of 50 μE.m-2.s-1 and at 22°C, plants were transferred to new plates containing 20 mM KNO3 and 20 mM NH4NO3 (referred here as 1xN: concentrations in MS media) in the absence or presence of light for 2 h at the start of their light cycle.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Mariana Obertello

PROVIDER: E-GEOD-21601 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Modeling the global effect of the basic-leucine zipper transcription factor 1 (bZIP1) on nitrogen and light regulation in Arabidopsis.

Obertello Mariana M Krouk Gabriel G Katari Manpreet S MS Runko Suzan J SJ Coruzzi Gloria M GM

BMC systems biology 20100812

<h4>Background</h4>Nitrogen and light are two major regulators of plant metabolism and development. While genes involved in the control of each of these signals have begun to be identified, regulators that integrate gene responses to nitrogen and light signals have yet to be determined. Here, we evaluate the role of bZIP1, a transcription factor involved in light and nitrogen sensing, by exposing wild-type (WT) and bZIP1 T-DNA null mutant plants to a combinatorial space of nitrogen (N) and light ...[more]

PMID: 20704717

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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

Expression data from Arabidopsis thaliana

Publications

Modeling the global effect of the basic-leucine zipper transcription factor 1 (bZIP1) on nitrogen and light regulation in Arabidopsis.

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