Project description:How developmental signals program gene expression in space and time is still poorly understood. Here, we addressed this question for the plant master regulator, auxin. Transcriptional responses to auxin rely on a large multigenic transcription factor family, the auxin response factors (ARFs). We deconvoluted the complexity of ARF-regulated transcription using auxin-inducible synthetic promoters built from cis-element pair configurations differentially bound by ARFs. We demonstrate using cellular systems that ARF transcriptional properties are not only intrinsic but also depend on the cis-element pair configurations they bind to, thus identifying a bi-layer ARF/cis-element transcriptional code. Auxin-inducible synthetic promoters were expressed differentially in planta showing at single-cell resolution how this bi-layer code patterns transcriptional responses to auxin. Combining cis-element pair configurations in synthetic promoters created distinct patterns, demonstrating the combinatorial power of the auxin bi-layer code in generating diverse gene expression patterns that are not simply a direct translation of auxin distribution.
Project description:Auxin orchestrates multiple transcriptional programs throughout plant development. In flowering plants, responses to auxin relies on a large multigenic family of transcription factors, the auxin response factors (ARF). While ARF combinations are thought to allow programming specific auxin target gene expression, this hypothesis has not been extensively tested. Here, we used a synthetic-driven reductionist approach to deconvolute the complexity of the ARF-dependent signaling. Synthetic promoters built from cis-elements identified as ARF preferential binding sites allowed us to demonstrate that ARF transcriptional properties are not intrinsic but depend on the cis-elements they bind to. Both in isolated cells and in planta, we show that specificity in transcription is encoded by both combinations of ARFs and of the cis-elements they target. Our results suggest that non-linear interactions between these two control layers constitute a unique regulatory system that can generate a wide diversity of gene expression patterns from the spatial auxin distribution.
Project description:Background: The auxin herbicides 2,4-D and dicamba are commonly used for management of horseweed (Erigeron canadensis L, (syn: Conyza canadensis L)). Halauxifen-methyl is a new auxin herbicide and recently commercialized to control several broadleaf weed species under a range of sites and environmental conditions. While synthetic auxin herbicides have been used for over 70 years, the precise mode of action that leads to plant death has yet to be clearly characterized. As new chemical families are discovered and the use of these herbicides continues to increase, it is imperative to understand how synthetic auxin active ingredients work within the plant to cause an herbicidal effect. Results: At 1 hour after treatment (HAT), 48 genes were consistently upregulated across the three herbicides, many of which are involved in the auxin-activated signaling pathway and response to auxin. At 6 HAT, 735 genes were upregulated by all herbicide treatments including genes associated with hormone signaling, metabolism, transport, senescence, and gene expression. The GO terms representing the 501 genes downregulated in all herbicide treatments were broadly categorized under two major groups: ATP and photosynthesis. At 6 HAT, over 50% of the genes differential expressed among the three herbicide treatments were unique to a single active ingredient. Conclusion: This research presents a first look into the differential gene expression profiles in horseweed following a foliar application of synthetic auxin compounds that represent three unique chemical families. While there are an abundance of transcriptome similarities induced by each herbicide that accounts for the general auxin herbicide response, distinct gene expression changes exclusive to each compound cannot be ignored as a contributor to the mode of action.
Project description:Mitochondrial stress stimuli such as AA caused a transient suppression of auxin signaling and conversely, auxin treatment represses a part of the response to AA treatment. Expression data of Col:LUC Arabidopsis treated with antimycin A (AA) in the presence or absence of a synthetic auxin analogue
Project description:The high metabolic flux through photorespiration constitutes a significant part of the carbon cycle. Although, the major enzymatic steps of the photorespiratory pathway are well characterized, little information is available on the functional significance of photorespiration beyond carbon recycling. Particularly important in this respect is the peroxisomal catalase activity which removes photorespiratory H2O2 generated during the oxidation of glycolate to glyoxylate, thus maintaining the cellular redox homeostasis governing the perception, integration and execution of stress responses. By perfroming a chemical screen, we identified 34 small molecules that alleviate the negative effects of photorespiration in Arabidopsis thaliana mutants lacking photorespiratory catalase (cat2). The chlorophyll fluorescence parameter photosystem II maximum efficiency (Fv'/Fm') was used as a high-throughput readout. The most potent chemical that could rescue the photorespiratory phenotype of cat2 is a pro-auxin that contains a synthetic auxin-like substructure belonging to the phenoxy herbicide family which can be released in planta. The naturally occurring indole-3-acetic acid (IAA) and other chemically distinct synthetic auxins also inhibited the photorespiratory-dependent cell death in cat2 mutants, implying a role for auxin signaling in stress tolerance. We used global transcriptome profiling to characterize the effect of a novel pro-axin structure (2-(2,4-dichlorophenoxy)-N-[4-(isobutyrylamino)-3-methoxyphenyl]propanamide) on Arabidopsis Col0 seedlings 2-(2,4-dichlorophenoxy)-N-[4-(isobutyrylamino)-3-methoxyphenyl]propanamid (5 μM) was added to seven-day-old Arabidopsis Col0 seedlings grown in a 96-well plate setup. Control plants were treated with solvent (DMSO) only. RNA was extracted 24 h following chemical addition.
Project description:The high metabolic flux through photorespiration constitutes a significant part of the carbon cycle. Although, the major enzymatic steps of the photorespiratory pathway are well characterized, little information is available on the functional significance of photorespiration beyond carbon recycling. Particularly important in this respect is the peroxisomal catalase activity which removes photorespiratory H2O2 generated during the oxidation of glycolate to glyoxylate, thus maintaining the cellular redox homeostasis governing the perception, integration and execution of stress responses. By perfroming a chemical screen, we identified 34 small molecules that alleviate the negative effects of photorespiration in Arabidopsis thaliana mutants lacking photorespiratory catalase (cat2). The chlorophyll fluorescence parameter photosystem II maximum efficiency (Fv'/Fm') was used as a high-throughput readout. The most potent chemical that could rescue the photorespiratory phenotype of cat2 is a pro-auxin that contains a synthetic auxin-like substructure belonging to the phenoxy herbicide family which can be released in planta. The naturally occurring indole-3-acetic acid (IAA) and other chemically distinct synthetic auxins also inhibited the photorespiratory-dependent cell death in cat2 mutants, implying a role for auxin signaling in stress tolerance. We used global transcriptome profiling to characterize the effect of a novel pro-axin structure (2-(2,4-dichlorophenoxy)-N-[4-(isobutyrylamino)-3-methoxyphenyl]propanamide) on Arabidopsis Col0 seedlings
Project description:Mitochondrial stress stimuli such as AA caused a transient suppression of auxin signaling and conversely, auxin treatment represses a part of the response to AA treatment. Expression data of Col:LUC Arabidopsis treated with antimycin A (AA) in the presence or absence of a synthetic auxin analogue Total RNA extraction was carried out on 80 mg of Col:LUC Arabidopsis seedlings grown on B5 media for 10 days and transferred to fresh plates, containing either B5 media or B5 media supplemented with 4.5 µM NAA for an additional 3 days. Seedlings were treated with 50 µM AA for 3 hours.
Project description:This study investigates the impact of 2 gene products (RBBP4, CHD1) on transcriptional programming in growing hiPSC at 0, 6 & 24 hours after acute protein depletion. Crispr editing was used to add an auxin-dependent degron to the C-termini of gene products expressed from the endogeneous loci and to insert an auxin-responsive degradation E3 ligase to the AAVS locus. Rapid (<30 min), specific target protein degradation in KOLF2.2J hiPSCs was induced by a synthetic auxin derivative (5-Ad-IAA) and bulk RNA-seq was performed at timepoints to define early (6 hr) and later (24 hr) downstream transcriptional dependencies.
Project description:mRNA and protein abundance are defined by transcriptional and post-transcriptional regulatory mechanisms. Here, we develop a machine learning pipeline, termed SONAR, to decipher the endogenous sequence code that determines mRNA and protein abundance in human cells. SONAR models predict up to 62% of mRNA and 63% of protein abundance independent of promoter or enhancer information, and reveal a strong—yet dynamic—cell-type specific sequence code. We also find that the effect of sequence features is dependent on their location within the mRNA transcript. Using SONAR, we design synthetic 3’UTRs, with which protein expression levels can be manipulated and tailored to a specific cell-type. Beyond its fundamental findings, our work provides novel means to improve immunotherapies and biotechnology applications.