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Positive fluorescent selection permits precise, rapid and in-depth over-expression analysis in plant protoplasts

ABSTRACT: Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the analysis of numerous aspects of plant biology. One drawback in the analysis of transformed protoplast suspensions is that they are a heterogeneous mix of cells that have and have not been successfully transfected. To overcome this problem, we have developed a system that employs a fluorescent positive selection marker in combination with flow cytometric analysis as well as fluorescence activated cell sorting (FACS) to isolate responses in the transfected protoplasts exclusively. This recombinase-compatible system enables high-throughput screening of genetic circuitry. Moreover, the use of FACS allows in depth downstream analysis. Lastly, over-expression is an effective means to dissect regulatory networks, especially where redundancy exists. Here, this system has been applied to the study of auxin signaling in order to investigate reporter gene activation and genome-wide transcriptional changes in response to manipulation of the auxin-response network. We have transiently over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors (IAA7mII and IAA19mII; Tiwari et al., 2001) in Arabidopsis Pwer::GFP root protoplasts, making use of a RFP fluorescent positive selection marker and FACS to isolate the dually labeled (IAAnmII expressing and Pwer::GFP-positive) cells. We have compared the transcriptional differences between an empty vector control, IAA7mII and IAA19mII protoplasts that had either been treated with 5microM IAA or mock-treated for 3 hours. Keywords: hormone treatment, genetic modification

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE13783 | GEO | 2009/03/01

SECONDARY ACCESSION(S): PRJNA110613

REPOSITORIES: GEO

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Transcription profiling of Arabidopsis Pwer::GFP protoplasts with over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors

Project description:Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the analysis of numerous aspects of plant biology. One drawback in the analysis of transformed protoplast suspensions is that they are a heterogeneous mix of cells that have and have not been successfully transfected. To overcome this problem, we have developed a system that employs a fluorescent positive selection marker in combination with flow cytometric analysis as well as fluorescence activated cell sorting (FACS) to isolate responses in the transfected protoplasts exclusively. This recombinase-compatible system enables high-throughput screening of genetic circuitry. Moreover, the use of FACS allows in depth downstream analysis. Lastly, over-expression is an effective means to dissect regulatory networks, especially where redundancy exists. Here, this system has been applied to the study of auxin signaling in order to investigate reporter gene activation and genome-wide transcriptional changes in response to manipulation of the auxin-response network. We have transiently over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors (IAA7mII and IAA19mII; Tiwari et al., 2001) in Arabidopsis Pwer::GFP root protoplasts, making use of a RFP fluorescent positive selection marker and FACS to isolate the dually labeled (IAAnmII expressing and Pwer::GFP-positive) cells. We have compared the transcriptional differences between an empty vector control, IAA7mII and IAA19mII protoplasts that had either been treated with 5microM IAA or mock-treated for 3 hours. Experiment Overall Design: 18 samples with 3 replicates for each condition and transformation vector: 3x empty vector mock treated, 3x empty vector IAA treated, 3x IAA7mII over-expressor mock treated, 3x IAA7mII over-expressor IAA treated, 3x IAA19mII over-expressor mock treated and 3x IAA19mII over-expressor IAA treated.

2009-03-07 | E-GEOD-13783 | biostudies-arrayexpress

AtNIGT1/HRS1 TARGET approach

Project description:This work studies the impact of AtNIGT1/HRS1-GR entrance in the nucleus upond DEX treatment in protoplasts. AtNIGT1/HRS1 TARGET. The whole procedure has been performed as previously described in Bargmann et al Mol Plant 2013. In brief, the protoplasts were transfected with the plasmid pBeaconRFP_GR-HRS1 that trigger the expression of HRS1 protein fused with the glucocorticoid receptor under control of CaMV35S promoter. Protoplasts were treated with 35µM cycloheximide (CHX) to inhibit translation and to select only direct target genes and 10µM dexamethasone (DEX) to induce HRS1-GR entry in the nucleus. Nitrate is maintained during the whole TARGET procedure. The Red Fluorescent Protein was used as marker selection for fluorescent-activated cell sorting (FACS) of successfully transformed protoplasts. RNA were extracted and amplified in order to be tested with ATH1 Affymetrix™ chips.

2015-01-05 | E-GEOD-64352 | biostudies-arrayexpress

Cell type-specific auxin responses in the Arabidopsis thaliana root

Project description:We performed an analysis of transcriptomic responses to auxin within four distinct tissues of the Arabidopsis thaliana root. This high-resolution dataset shows how different cell types are predisposed to react to auxin with discrete transcriptional responses. The sensitivity provided by the analysis lies in the ability to detect cell-type specific responses diluted in organ-level analyses. This dataset provides a novel resource to examine how auxin, a widespread signal in plant development, influences differentiation and patterning in the plant through tissue-specific transcriptional regulation. To analyze the effect of auxin in separate spatial domains of the root, early transcriptional changes in response to auxin treatment were assayed by means of fluorescence activated cell sorting (FACS) and microarray analysis in four tissues of the Arabidopsis root (wild type Col-0). The samples covered inner and outer as well as proximal and distal cell populations; including the stele (reporter line pWOL::GFP), xylem-pole (xp) pericycle (enhancer trap line E3754), epidermis/lateral root cap (reporter line pWER::GFP) and columella (enhancer trap line PET111). One-week-old seedlings of the individual lines were treated with auxin (two hours, 5µM indole-3-acetic acid [IAA]) or mock treated, after which roots were harvested and cells were dissociated by cell wall digestion (1 hour; including 5uM IAA) . GFP-positive cells were sorted and used for microarray transcriptome analysis (as in Bargmann and Birnbaum, Plant Phys. 2010). For comparison, transcriptional responses to auxin were also assayed in intact (undigested) roots.

2013-08-06 | E-GEOD-35580 | biostudies-arrayexpress

4sU-Seq of human U2OS cells upon acute RBM8A protein depletion

Project description:The impact of acute RBM8A depletion on nascent transcription (using the auxin (IAA)-inducible degron system in conjunction with 4sU-sequencing) was investigated.

2022-07-13 | GSE207898 | GEO

Variation in auxin sensing guides AUX/IAA repressor ubiquitylation and destruction

Project description:Auxin is a drug-like small molecule and morphogen that triggers the formation of SCFTIR1/AFB-AUX/IAA co-receptor complexes leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors in plants. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. TIR1-IAA19 and TIR1-IAA6 interactions form co-receptors with different affinities, which specify ubiquitylation and turnover rate of the AUX/IAA. We demonstrate lysine ubiquitylation in IAA6/IAA19 and propose this ubiquitylation signature to be a consequence of auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologs, that contributes differentially to auxin sensing. We postulate that AUX/IAAs have emerged as a versatility toolbox for auxin-modulated transcriptional control, as the gamut of AUX/IAA destruction kinetics enables fine-tuning of transcriptional auxin response and contributes to the complexity of hormone signaling.

2017-06-14 | PXD004027 | Pride

Expression profiling of genes involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton

Project description:We used a next-generation sequencing approach, Illumina Digital Gene Expression (DGE) technology, to Genome-wide profiling of gene expression during cotton SE. As a result, 5,076 differentially expressed genes were identified during cotton SE. Expression profile and functional assignments of these genes indicated significant transcriptional complexity during this process. Transcription factor-encoding genes were found to be differentially regulated during SE. The complex pathways of auxin abundance, transport and response with differentially regulated genes revealed that the auxin-related transcripts belonged to IAA biosynthesis, indole-3-butyric acid (IBA) metabolism, IAA conjugate metabolism, auxin transport, auxin-responsive protein / indoleacetic acid-induced protein (Aux/IAA), auxin response factor (ARF), small auxin-up RNA (SAUR), Aux/IAA degradation, and other auxin-related proteins, which allow an intricate system of auxin utilization to achieve multiple purposes in SE. Quantitative real-time PCR (qRT-PCR) was performed on selected genes with different expression patterns and functional assignments were made to demonstrate the utility of RNA-Seq for gene expression profiles during cotton SE. We report here the first comprehensive analysis of transcriptome dynamics that may serve as a gene expression profile blueprint in cotton SE. Our main goal was to adapt the RNA-Seq technology to this notable development process and to analyse the gene expression profile. Complex auxin signalling pathway and transcription regulation were highlighted. Together with biochemical and histological approaches, this study provides comprehensive gene expression data sets for cotton SE that serve as an important platform resource for further functional studies in plant embryogenesis. differential gene expression analysis at 9 time-points/stages during somatic embryogenesis in cotton by deep sequencing, using Illumina GAIIx.

2012-05-31 | E-GEOD-38209 | biostudies-arrayexpress

Auxin mediated gene expression in WT, arf7, arf19 and arf7 arf19 mutants

Project description:RNA samples were extracted from liquid cultured seedlings treated with or without auxin (5ÂµM IAA) for 2 h. Lines used for this study: Columbia wild-type nph4-1(arf7) single mutant arf19-1 single mutant nph4-1 arf19-1 double mutant Treatment: Control (EtOH) Auxin treated (5ÂµM IAA) Keywords = Auxin Keywords = Auxin response factor Keywords: parallel sample

2010-06-09 | E-GEOD-627 | biostudies-arrayexpress

Auxin mediated gene expression in WT, arf7, arf19 and arf7 arf19 mutants

Project description:RNA samples were extracted from liquid cultured seedlings treated with or without auxin (5µM IAA) for 2 h. Lines used for this study: Columbia wild-type nph4-1(arf7) single mutant arf19-1 single mutant nph4-1 arf19-1 double mutant Treatment: Control (EtOH) Auxin treated (5µM IAA) Keywords = Auxin Keywords = Auxin response factor Keywords: parallel sample

2005-10-19 | GSE627 | GEO

Grigolon2018 - Responses to auxin signals

Project description:Plants depend on the signalling of the phytohormone auxin for their development and for responding to environmental perturbations. The associated biomolecular signalling network involves a negative feedback on Aux/IAA proteins which mediate the influence of auxin (the signal) on the auxin response factor (ARF) transcription factors (the drivers of the response). To probe the role of this feedback, we consider alternative in silico signalling networks implementing different operating principles. By a comparative analysis, we find that the presence of a negative feedback allows the system to have a far larger sensitivity in its dynamical response to auxin and that this sensitivity does not prevent the system from being highly resilient. Given this insight, we build a new biomolecular signalling model for quantitatively describing such Aux/IAA and ARF responses.

2022-05-10 | MODEL2003060002 | BioModels

Grigolon2018-Responses to auxin signals

2021-09-19 | BIOMD0000000927 | BioModels

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