A Kinetic Analysis of Auxin-mediated Changes in Transcript Abundance in Arabidopsis Reveals New Mediators of Root Growth and Development
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ABSTRACT: Auxin-dependent transcript abundance was assayed by transferring 6 day old Arabidopsis grown on a a nylon mesh to IAA-containing or control media
Project description:Auxin-dependent transcript abundance was assayed by transferring 6 day old Arabidopsis grown on a a nylon mesh to IAA-containing or control media Seedling roots were harvested 0, 0.5, 1, 2, 4, 8, 12, or 24 hours after treatment and the resultant RNA was used for microarray analysis to determine the kinetic profiles of auxin-responsive gene expression. 8 timepoints after control or IAA treatment done in triplicate
Project description:Ethylene-dependent gene expression was assayed by treating with 1 uM ACC, an ethylene precursor, or a control treatment to Arabidopsis seedlings by transferring 6 day old Arabidopsis grown on a a nylon mesh to fresh ACC-containing or control media Seedling roots were harvested 0, .5, 1, 2, 4, 8, 12, and 24 hours after treatment and the resultant RNA was used for microarray analysis to determine the kinetic profiles of auxin-responsive gene expression
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.
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.
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
Project description:Cullin RING-type E3 ubiquitin ligase SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface- exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.
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
Project description:To assess natural variation of downstream auxin responses we subjected 7 different arabidopsis ecotypes to a time course of auxin treatments. 7d-old seedlings grown in liquid culture have been treated for 0, 30 min, 1h and 3h with 1 µM IAA. Experiment Overall Design: 83 samples were used in this experiment
Project description:Chemical signaling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via a novel auxin degradation locus was essential for maintaining stereotypic root development in an ecologically-relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon’s MarR-family repressor with IAA and other auxins. We identify auxin-degradation operons across the bacterial tree of life and define two distinct types based on gene content and metabolic products: iac-like and iad-like. We solve the structures of MarRs from representatives of each auxin degradation operon type, establishing that each have distinct IAA binding pockets. Comparison of representative IAA degrading strains from diverse bacterial genera show that while all degrade IAA, only strains containing iad-like auxin degrading operons interfere with auxin signaling in a complex synthetic community context. This suggests that iad-like operon containing strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome.
Project description:To assess natural variation of downstream auxin responses we subjected 7 different arabidopsis ecotypes to a time course of auxin treatments. 7d-old seedlings grown in liquid culture have been treated for 0, 30 min, 1h and 3h with 1 µM IAA. Keywords: Expression profilling by array