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.

• MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• kinetika MeSH
• proteomika metody   MeSH
• pyl metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny chemie   metabolismus   MeSH
• tabák genetika   metabolismus   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.

• MeSH
• 2D gelová elektroforéza MeSH
• chromatografie afinitní metody   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfoproteiny analýza   chemie   izolace a purifikace   MeSH
• fosforylace MeSH
• molekulární sekvence - údaje MeSH
• proteom analýza   chemie   MeSH
• pyl chemie   MeSH
• rostlinné proteiny analýza   chemie   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• tabák chemie   MeSH
• titan MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH