Project description:Here we report proteins identified after conducting Tandem Affinity Purification (TAP) of the TOPLESS (TPL) corepressor from Arabidopsis. We generated transgenic plants harboring TPL fused to the GS-TAG, "Boosting tandem affinity purification of plant protein complexes" (Van Leene et al., 2008) [1]. Four independent biological replicates of a selected TPL-GS-TAG line were grown simultaneously, crosslinked with formaldehyde, and proteins were isolated from whole plant tissue via TAP. Purified proteins were treated with trypsin, and the peptides were analyzed via mass spectrometry. Datasets are hosted in the MassIVE public repository (reference number: MSV000082477, https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=f16255fb7080426a9fe1926b4d3d5862). The data in this article has not been published elsewhere and is original to this work.
Project description:To identify Arabidopsis Wdr8 interactors, immunoprecipitation associated LC-MS/MS analysis was carried out. Crude proteins extracted from Wdr8-GFP expressing transgenic plants was subjected to the immuno-precipitation assay using GFP antibody magnetic beads (µMACS GFP isolation kit, Miltenyi Biotec). Co-purified proteins were separated by 10% SDS-PAGE gel and stained with SYPRO Ruby (BioRad laboratories). The stained protein bands were excised into several fraction pieces according to protein sizes, and in-gel protein digestion by trypsin was carried out. Purified protein peptides were subjected to LC-MS/MS analysis (LTQ-Orbitrap XL-HTC-PAL system). Collected MS/MS peak spectra was analyzed by the MASCOT server to identify peptide sequence.
Project description:Numerous gene expression datasets from diverse tissue samples from the plant variety Arabidopsis thaliana have been already deposited in the public domain. There have been several attempts to do large scale meta-analyses of all of these datasets. Most of these analyses summarize pairwise gene expression relationships using correlation, or identify differentially expressed genes in two conditions. We propose here a new large scale meta-analysis of all of the publicly available Arabidopsis datasets to identify Boolean logical relationships between genes. Boolean logic is a branch of mathematics that deals with two possible values. In the context of gene expression datasets we use qualitative high and low expression values. A strong logical relationship between genes emerges if at least one of the quadrants is sparsely populated. We put together a web resource where gene expression relationships can be explored online which helps visualize the logical relationships between genes. We believe that this website will be useful in identifying important genes in different biological context. The web link is http://hegemon.ucsd.edu/plant/.
Project description:Interactors of the plant natriuretic peptide present in Arabidopsis thaliana, termed AtPNP-A, were affinity-based isolated from A. thaliana (Col-0) leaf mesophyll cell protoplasts by incubating the protoplasts with biologically active biotinylated peptide corresponding to amino acid sequence of the active site of AtPNP-A (pAtPNP-A), either in the presence or absence of a cross-linking agent, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), or with equimolar amount of biotin with DTSSP (negative control). Upon biotin/streptavidin-based isolation of proteins bound to the pAtPNP-A or biotin, the proteins were separated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), digested with trypsin and subjected to identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Label-free quantification of identified proteins allowed identification of binding partners of AtPNP-A, paving the way for pinpointing novel signal transduction pathways AtPNP-A is involved in. The raw and processed LC-MS/MS data reported in this article have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD017925.
Project description:Rab GTPases are key spatio-temporal regulators of endomembrane trafficking and endomembrane compartment identity across eukaryotes. This therefore makes them ideal targets for study of endomembrane compartment and trafficking pathway composition. In plants, few definitive interactors of Rab GTPases and cargoes of their associated trafficking pathways are known, hindering our understanding of how plants may accomplish and support some unique aspects and requirements of their post-Golgi trafficking systems. The purpose of this work was to identify candidate interactors of different post-Golgi Rab GTPases in Arabidopsis. Co-IPs were performed against tagged-versions of three different post-Golgi Rab GTPases: RAB-A5c, RAB-A2a and RAB-G3f, as well as against control wild-type (Col-0) plants. Comparative ranking of the different proteomes produced candidate interactor lists enriched in possible specific interactors of each Rab GTPase, as well as generic shared interactors. This dataset forms the basis of the characterisation work of several upcoming publications.
Project description:The goal of this study was to identify small RNA-ARGONAUTE interactions in Arabidopsis. Co-immunoprecipitation assays followed by high-throughput sequencing were done to identify small RNA associated with HA-AGO2 and HA-AGO7. Experiment Overall Design: Sequencing-by-synthesis technology was used to sequence small RNA from input and co-immunoprecipitation fractions from cell lysates of inflorescence tissue from Arabidopsis plants transformed with HA epitope-tagged AGO2 or AGO7 constructs. Meaningful intepretation requires comparison between the input and co-immunoprecipitation datasets. Experiment Overall Design: Note: Raw data files requested, however, the submitter declined to provide them.
Project description:In order to identify the comprehensive composition of Arabidopsis mitochondrial ribosome proteins, a strategy based on complementary approaches was used. Classical biochemical purification of ribosomes was combined with the immunoprecipitation of mitoribosomes using a specific Arabidopsis mitoribosome protein as a bait and quantitative proteomics. The immuno-purifications (IP) of the mitoribosome were performed on purified mitochondria from rPPR1-HA plant line (flowers as starting material). IPs were performed performed in different conditions (i.e. with 100, 400 or 600 mM KCl at the IP washing step or 800mM all along the IP). For each condition, experiments were performed in triplicates and IP proteins were identified by quantitative nano LC-ESI-MS/MS. Classic biochemical purification of mitochondrial monosomes (i.e. free ribosomes) was also performed. Starting from purified wild-type Arabidopsis mitochondria (cell culture as starting material), ribosomes were separated on high resolution 10-30% continuous sucrose gradients and mitoribosome containing fractions were analyzed by quantitative nano LC-ESI-MS/MS.
Project description:Photoperiodic floral initiation in the leaf is controlled by the hub gene CONSTANS (CO) while jasmonates (JAs) control flower senescence. Although both processes are chronologically ordered, no association between them has been described to date. We show that CO protein remains in Arabidopsis flowers after the floral induction, although displaying a different tissue and diurnal pattern than in leaves. We found that changes in CO expression alter flower senescence and abscission by interfering with the JA response, supported by petal specific analysis as well as CO overexpression in JA synthesis and signaling mutants. CO has a ZIM-like domain that mediates interaction with the JA response repressor JAZ3 (jasmonate ZIM-domain 3), inhibiting its repressor activity and activating downstream transcription factors involved in flower senescence. The complex CO-JAZ3 also interacts with the E3 ubiquitin ligase Coronatine Insensitive 1 (COI1) leading to its degradation in the presence of jasmonates. Therefore, the coordinated recruitment of photoperiodic and jasmonate signaling pathways would be an efficient way for plants to chronologically order the floral process and ensure the success of offspring production.
Project description:We found that USF2 is repressor of lysosomal genes. To find the interacting co-repressor complex of USF2, we performed USF2 complex purification and mass spectrography.