title: Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.
abstract: 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, because 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.
authors.affiliation: From the ‡Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, v.v.i., Rozvojova 263, 165 00 Praha 6, Czech Republic;
authors.forename: Jan
authors.initials: J
authors.lastname: Fíla
authors.affiliation: §Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Straβe 6b, 44227 Dortmund, Germany;
authors.forename: Sonja
authors.initials: S
authors.lastname: Radau
authors.affiliation: ¶Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetic and Crop Plant Research, Corrensstraβe 3, 06466 Gatersleben, Germany;
authors.forename: Andrea
authors.initials: A
authors.lastname: Matros
authors.affiliation: ¶Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetic and Crop Plant Research, Corrensstraβe 3, 06466 Gatersleben, Germany;
authors.forename: Anja
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: ‖Department of Breeding Research, Leibniz Institute of Plant Genetic and Crop Plant Research, Corrensstraβe 3, 06466 Gatersleben, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Scholz
authors.affiliation: From the ‡Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, v.v.i., Rozvojova 263, 165 00 Praha 6, Czech Republic;
authors.forename: Jana
authors.initials: J
authors.lastname: Feciková
authors.affiliation: ¶Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetic and Crop Plant Research, Corrensstraβe 3, 06466 Gatersleben, Germany;
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Mock
authors.affiliation: From the ‡Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, v.v.i., Rozvojova 263, 165 00 Praha 6, Czech Republic;
authors.forename: Věra
authors.initials: V
authors.lastname: Čapková
authors.affiliation: §Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Straβe 6b, 44227 Dortmund, Germany;
authors.forename: René Peiman
authors.initials: RP
authors.lastname: Zahedi
authors.affiliation: From the ‡Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, v.v.i., Rozvojova 263, 165 00 Praha 6, Czech Republic; david@ueb.cas.cz.
authors.forename: David
authors.initials: D
authors.lastname: Honys
mesh_terms: D001665:Binding Sites; D018506:Gene Expression Regulation, Plant; D007700:Kinetics; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011058:Pollen / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D010940:Plant Proteins