Dataset Information

Pollen proteome map of Arabidopsis thaliana

ABSTRACT: Not available

INSTRUMENT(S): instrument model, LTQ

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Pollen

SUBMITTER: Katja Baerenfaller

PROVIDER: PRD000084 | Pride | 2012-01-31

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_15505.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_15505.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_15505.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_8743.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_8743.pride.mztab.gz	Mztab

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Publications

Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function.

Grobei Monica A MA Qeli Ermir E Brunner Erich E Rehrauer Hubert H Zhang Runxuan R Roschitzki Bernd B Basler Konrad K Ahrens Christian H CH Grossniklaus Ueli U

Genome research 20090622 10

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously ...[more]

PMID: 19546170

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Project description:Plant Growth and Treatment. Seedlings of Arabidopsis thaliana GVG::FLAG-NtMEK2DD were grown in 50 ml of half-strength Murashige and Skoog medium at 22°C under continuous light (70 µE/m2/sec). Twelve-day-old seedlings were treated with DEX (1 µM) or ethanol (control) and collected 6 h after treatment. Protein Extraction and MOAC Enrichment of Phosphoproteins. Protein extraction from seedlings and enrichment of phosphoprotein. The concentration of protein in extracts was determined using the Bio-Rad protein assay kit with BSA as the standard. Western Blotting Analysis and Immunodetection. Protein samples were subjected to SDS-PAGE, transferred to nitrocellulose, and used for immunodetection. Mouse monoclonal anti-Flag M2 antibodies were purchased from Sigma-Aldrich; all other antibodies were from New England Biolabs. Chemiluminescence detection of antigen-antibody complexes was done with Immobilon™ Western substrate (Millipore). Enrichment of Phosphopeptides by MOAC. For in-solution protein digestion, total proteins or MOAC-enriched phosphoproteins were predigested for 5 h with endoproteinase Lys-C (1/100 w/w) followed by trypsin digestion (Poroszyme immobilized trypsin (1/100 v/w)) overnight. Protein digests were desalted using a SPEC C-18 96-well plate (Varian) according to the manufacturer’s instructions. TiO2 beads were purchased from Glygen Inc. Mass Spectrometry and Protein Identification. Peptides were analyzed using an Orbitrap XL mass spectrometer (Thermo Scientific). Chromatography was performed using a Chromolith CapRod C18 monolithic column with 0.1% (v/v) formic acid (FA) in ultrapure H2O and 0.1% (v/v) FA in 90% (v/v) acetonitrile (ACN) in ultrapure H20 as mobile phases and a gradient from 10% to 35% organic phase in 120 min with a flow rate of 500 nl/min. Multistage activation/pseudoMS3 was enabled for acquisition of CID tandem MS (MS/MS) mass spectra of phosphopeptides with a neutral loss mass list of 97.97, 48.999, 32.66 and 24.49 Da. Database search was performed with the Proteome Discoverer Software version 1.2. using the SEQUEST algorithm.

Project description:BACKGROUND: To understand transcriptional regulatory networks (TRNs), especially the coordinated dynamic regulation between transcription factors (TFs) and their corresponding target genes during development, computational approaches would represent significant advances in the genome-wide expression analysis. The major challenges for the experiments include monitoring the time-specific TFs' activities and identifying the dynamic regulatory relationships between TFs and their target genes, both of which are currently not yet available at the large scale. However, various methods have been proposed to computationally estimate those activities and regulations. During the past decade, significant progresses have been made towards understanding pollen development at each development stage under the molecular level, yet the regulatory mechanisms that control the dynamic pollen development processes remain largely unknown. Here, we adopt Networks Component Analysis (NCA) to identify TF activities over time course, and infer their regulatory relationships based on the coexpression of TFs and their target genes during pollen development. RESULTS: We carried out meta-analysis by integrating several sets of gene expression data related to Arabidopsis thaliana pollen development (stages range from UNM, BCP, TCP, HP to 0.5 hr pollen tube and 4 hr pollen tube). We constructed a regulatory network, including 19 TFs, 101 target genes and 319 regulatory interactions. The computationally estimated TF activities were well correlated to their coordinated genes' expressions during the development process. We clustered the expression of their target genes in the context of regulatory influences, and inferred new regulatory relationships between those TFs and their target genes, such as transcription factor WRKY34, which was identified that specifically expressed in pollen, and regulated several new target genes. Our finding facilitates the interpretation of the expression patterns with more biological relevancy, since the clusters corresponding to the activity of specific TF or the combination of TFs suggest the coordinated regulation of TFs to their target genes. CONCLUSIONS: Through integrating different resources, we constructed a dynamic regulatory network of Arabidopsis thaliana during pollen development with gene coexpression and NCA. The network illustrated the relationships between the TFs' activities and their target genes' expression, as well as the interactions between TFs, which provide new insight into the molecular mechanisms that control the pollen development.