Proteomic characterization of plasma membrane-enriched fraction from tobacco (Nicotiana tabacum L. cv Samsun) pollen tubes.
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Nicotiana Tabacum (common Tobacco)
TISSUE(S): Plant Cell, Pollen
DISEASE(S): Disease Free
SUBMITTER: 
Martin Hubalek
LAB HEAD: Martin Hubálek
PROVIDER: PXD037046 | Pride | 2022-10-27
REPOSITORIES: Pride
Similar Datasets
Project description:Transcriptome profiling of three developmental stages of immature male gametophyte intobacco (Nicotiana tabacum) Total RNA isolated from tobacco microspores and early and late bicellular pollen was hybridised on Agilent Tobacco Gene Expression Microarray 4x44K in two biological replicates per sample
2014-10-16 | E-GEOD-62349 | biostudies-arrayexpress
Project description:Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, since some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.
2016-01-27 | PXD003042 | Pride
Project description:Mitogen activated protein kinases (MAPKs) and phosphoinositides contribute to establishment and maintenance of polarity in eukaryotic cells. However, interactions between these important signaling pathways are largely unclear. Pollen tubes are an important model for the study of polarized cell expansion, in which PI4P 5-kinases control apical secretion and MAPKs contribute to pollen tube guidance. Here we report on the regulation of the Arabidopsis PI4P 5-kinase, AtPIP5K6, by the MAPK, MPK6. Purified recombinant AtPIP5K6 was phosphorylated in vitro upon incubation with pollen tube extracts. Non-targeted in-gel-kinase assays of pollen tube extracts followed by mass-spectrometry identified MPK6 as a candidate protein kinase upstream of AtPIP5K6. MPK6 interacted with AtPIP5K6 in yeast-two-hybrid tests, and bimolecular fluorescence complementation verified the interaction at the apical plasma membrane of pollen tubes. Recombinant AtPIP5K6 was phosphorylated in vitro by recombinant MPK6, resulting in reduced catalytic activity of AtPIP5K6. Alanine-substitution of phosphorylation sites, T590A and T597A, abrogated phosphorylation of AtPIP5K6 by MPK6 and prevented the in vitro inhibition. A T597D substitution resulted in reduced catalytic activity of the modified AtPIP5K6 protein in vitro. Coexpression of MPK6 with AtPIP5K6 in pollen tubes resulted in attenuation of PI4P 5-kinase-mediated polarity defects in vivo. Reciprocally, AtPIP5K6 effects were enhanced upon concomitant RNAi-inhibition of endogenous tobacco MAPKs. Both effects were absent when a AtPIP5K6 T590A T597A-variant was expressed, which could no longer be phosphorylated. We conclude that MPK6 limits PI4P 5-kinase-dependent polarized secretion to effect pollen tube guidance, revealing a new link between conserved signaling pathways with potential ramifications across eukaryotic kingdoms.
2022-02-22 | PXD006067 | Pride
Project description:This was a pilot project carried out by Dr Wojciech Majeran to determine the maize pollen proteome harvested from field-grown W22 (T43) plants in plots on the Musgrave Research Farm (Cornell CALS) in Aurora (NY). To enhance proteome coverage, the pollen were separated into soluble and membrane bound protein fractions, and separated by SDS-PAGE followed by in-gel digestion and shot-gun proteomics using a nanoLC-Orbitrap system.
2023-09-11 | PXD045274 | Pride
Project description:As in animals, cell-cell communication plays pivotal role in male-female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective. We perform genome-wide quantitative liquid chromatography coupled tandem mass spectrometry of a pistil-stimulated pollen tube secretome and identify 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube-secretome is dominated by unconventional-type secreted proteins representing 57% of the total secretome. In support, we show that unconventional-type protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discover that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that Arabidopsis thaliana translationally controlled tumor protein-knockdown plants have pollen tubes that poorly navigate to the target ovule, and the knocked down allele is poorly transmitted through the male. We show that regulators of the endoplasmic reticulum-trans-golgi network protein secretory pathway control secretion of pollen tube-secreted cysteine-rich proteins, including pollen tube attractants, and are essential for pollen tube growth and guidance, as well as ovule-targeting competence. This work, the first pollen tube secretome study, identifies novel genome-wide pollen tube-secreted proteins with potential function in pollen tube-ovule guidance for sexual reproduction. Functional analysis highlights a potential mechanism for pollen tube unconventional protein secretion and reveals likely regulators of pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggest secretion via exosomes as a possible mechanism for cell-cell communication between the pollen tube and female reproductive cells. For processed dataset with quantitative information, see Hafidh S, Potesil D, Fila J et al. Genome Bilogy 2016.
2016-03-18 | PXD002215 | Pride
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis. Experiment Overall Design: Pollen and pollen tubes were collected as described in the protocols section for RNA extraction and hybridization on Affymetrix ATH1 Genechip microarrays.
2009-08-15 | E-GEOD-17343 | biostudies-arrayexpress
Project description:We used tomato pollen in order to identify pollen stage-specific small non-coding RNAs (sncRNAs) and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing was performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions.
2015-08-20 | E-MTAB-3830 | biostudies-arrayexpress
Project description:The expression analysis had two goals: (1) look at relative transcription within mature pollen grains (2) compare expression in the stigma during pollination with either compatible or in-compatible pollen. Two pairwise comparisons, (i) unpollinated stigma vs. stigma pollinated with compatible pollen, and (ii) unpollinated stigma vs stigma pollinated with incompatible pollen. The genotype where stigma samples were harvested from is F1-30, and this is also the pollen source during an incompatible pollination reaction. The compatible pollen source is the variety Foxtrot (heterogeneous populations).
2015-10-26 | E-MTAB-3760 | biostudies-arrayexpress
Project description:Tomato pollen tube RNAseq data
2021-04-14 | E-MTAB-9725 | biostudies-arrayexpress
Project description:In sexual reproduction, a proper communication and cooperation between male and female organs and tissue are essential for male and female gametes to unite. In flowering plants, female sporophytic tissues and gametophyte direct a male pollen tube towards an egg apparatus, which consists of an egg cell and two synergid cells. The cell-cell communication between the pollen tube and the egg apparatus, such as the reception of a signal from the egg apparatus at the pollen tube, makes the tip of pollen tube rapture to release the sperm cell. To isolate male factors involved in the interaction between a pollen tube and an egg apparatus, we focused on receptor-like kinases (RLKs), which are extensively diversified in the flowering plant lineage to comprise a large monophyletic gene family. Approximately 620 members were found in the Arabidopsis thaliana genome. Expression patterns of 558 RLKs were analyzed using an Affymetrix ATH1 microarray of A. thaliana. We focused on two RLKs, ANXUR1 (ANX1) and ANXUR2 (ANX2), and characterized their function. Here we report that pollen tubes of anx1/anx2 ruptured before arriving at the egg apparatus, suggesting that ANX1 and ANX2 are male factors controlling pollen tube behavior with directing rupture at proper timing. Furthermore, ANX1 and ANX2 were the most closely related paralogs to a female factor FERONIA/SIRENE controlling pollen tube behavior expressed in synergid cells. Our finding shows that the coordinated behaviors of female and male reproductive apparatuses are regulated by the sister genes, whose duplication might play a role in the evolution of fertilization system in flowering plants. Experiment Overall Design: Arabidopsis thaliana Col-0 was grown at 18°C for 8 hr in the dark and at 22°C for 16 hr in the light. To collect germinated pollen with pollen tubes, opened flowers were touched to cellophane layered on the pollen germination medium containing 13.5% sucrose, 10 mM CaCl2, 0.4 mM boric acid, 1 mM KCl, 0.001% myo-inositol, and 0.6% agarose. The released pollen was incubated at 25°C for 6 hr in the dark. For collection of seedlings, seeds were sown on Murashige-Skoog medium containing 1x Murashige and Skoog Plant Salt Mixture (Wako), 1x Gamborgâs vitamin solution (Wako), 1% sucrose, and 0.8% agar. Seedlings including roots with one or two rosette leaves were collected for RNA extraction. RNA was extracted with Isogene (Nippon Gene), precipitated with LiCl, and further purified with RNeasy. The ATH1 genechip (Affymetrix) was used for expression analysis using 10 µg each of total RNA following the manufacturer's instructions. Array data were processed and analyzed with the GCOS1.2 program (Affymetrix) and Excel 2004 (Microsoft). Hybridization with germinating pollen RNA was duplicated and the results were compared to that with seedling RNA.
2009-08-09 | E-GEOD-12316 | biostudies-arrayexpress