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: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: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:The aim of the project is to compare NAA-induced proteome remodelling between wild-type plants (Arabidopsis thaliana, Col-0) to autophagy deficient mutants (atg2-1) treated MS growt media suplemented with solvent (EtOH- Control) or NAA (a synthetic variant of the plant hormone auxin. The aim is to look at the rapid response when treated with auxin and as so the treatment is very short at 30 minutes. The time of the experiment was at zeitgeber 0.
Project description:Wild tobacco flowers wave rhythmically to facilitate specific pollinator interactions. This movement behavior is controlled by a regulatory network that involves the circadian clock- and auxin-signaling pathways. The plant hormone auxin, similarly to its function in tropic movements, acts as growth regulator in the circadian regulation of floral movement. Dorsoventral asymmetry in auxin levels and auxin transcriptional responses mediate the growth responses in the floral peduncle that make flowers move. Multiple components of the auxin-signaling pathway and auxin responses are under the control of circadian clock. However, it is unclear where these two pathways intersect and how collectively contribute to regulate specific rhythmic outputs. Here we found that the blue light photoreceptor and circadian clock component ZEITLUPE (ZTL) controls auxin responses through the regulation of the auxin-signaling pathway in a time-of-day and blue light specific manner. Abrogation of ZTL expression abolishes flower movement and the temporal gating of auxin-induced growth responses in the floral peduncle. ZTL regulates transcription and directly interacts with indole-3-acetic acid inducible 19 (IAA19), a circadian controlled gene that regulates development of curvature in moving organs. Indicating that ZTL modulates auxin sensitivity in part through the regulation of AUX/IAA transcriptional repressors. At night, growth responses in the peduncle to the synthetic auxin 2,4-D revealed that ZTL additionally controls auxin responses regulating auxin homeostasis. These results indicate that ZTL conveys temporal and environmental information, at multiple levels, into the auxin signaling-pathway and in this way sculpts the temporal gating of auxin responses that allow flowers to move. To gain further insight into the molecular basis of temporal regulation of the movement of flowers we used a whole genome microarray as a discovery platform to identify genes differentially expressed in a RNAi knockdown line silenced in the expression of the circadian clock component ZEITLUPE (irZTL-314).
Project description:Combinatorial genetic perturbations have been utilized to identify synthetic sick/lethal genetic interactions for cancer drug target discovery. Current methods for high-throughput combinatorial genetic screening are inefficient and cumbersome. Here we developed a simple, robust, and high-performance CRISPR-Cas12a-based approach for unbiased, combinatorial genetic screening in cancer cells.
Project description:Endoplasmic reticulum (ER) is an intracellular extensive network of membranes. Among major ER functions belong proteosynthesis, protein folding, their post-transcriptional modification and sorting within the cell and last but not least lipid anabolism. Moreover, several recent studies have indicated involvement of plant ER in regulation of intracellular auxin homeostasis by modulation of its metabolism. Therefore, in order to study auxin metabolome in ER, it is necessary to obtain highly enriched and, if possible, pure ER fraction. The separation of ER is troublesome due to similar biochemical properties with other cellular endomembranes. The majority of published protocols for ER isolation utilise density gradient ultracentrifugation despite the suboptimal resolving power of this approach. This work aims to optimize the isolation of ER from Arabidopsis thaliana for the subsequent determination of ER-specific auxin metabolite profiles by mass spectrometry. Following auxin metabolite determination showed a high increase of active auxin form within ER compared to whole plant. Moreover, our optimized isolation of ER may be further followed by multiple “omics” technologies including analysis of macromolecular as well as low molecular-weight compound from same sample.