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.

Fig. 1.

A schematic workflow of the performed experiments. The three stages of tobacco male gametophyte (particularly mature pollen, pollen activated in vitro for 5 min and pollen activated in vitro for 30 min) were subjected to TCA/acetone protein extraction, trypsin digest and phosphopeptide enrichment by TiO₂. The obtained phosphopeptide-enriched eluate was fractionated by nLC and measured by MS/MS. The present phosphopeptides were identified (if possible with the unambiguous position of the phosphosite) and the results further analyzed.

Fig. 2.

The statistics of identified phosphopeptides. A, Column diagram showing the number of phosphopeptides according to the number of unambiguously identified phosphorylation sites in a single peptide. If a phosphorylation site was identified in more than one peptide, it was counted repeatedly every time on every peptide. B, Number of phosphorylation sites according to the phosphorylated amino acid (serine, threonine, or tyrosine) identified. If a phosphorylation site was identified in more than one peptide, it was counted only once.

Fig. 3.

Pie chart showing the percentage of the individual phosphoprotein categories.

Fig. 4.

Expression profiles of the selected phosphopeptides with a different abundance in the studied male gametophyte stages. The phosphopeptides were sorted into seven regulation groups based on their abundance differences in the three analyzed male gametophyte stages (group I - left panel; groups II-VII - right panel). The relative peptide abundance in each group is shown based on a gray scale (light gray - not detected; black - the highest concentration). Each column represents the average peptide abundance of the three independent LC-MS experiments. In the rows, the normalized abundance of peptides as extracted from the Proteome discoverer LC-MS software is presented. Peptides assigned to one and the same identifier are highlighted in gray. Gene ontology (GO) categories are presented for each group as a pie chart. The full presentation of the data set is provided in the supplemental Table S3.

Fig. 5.

Phosphorylation patterns of two selected phosphoproteins identified in our data set in three stages of male gametophyte. A, Actin cytoskeleton-regulatory complex protein PAN1-like. B, PB1-containing protein. “S” stands for serine, the number indicates the position of the amino acid in the polypeptide chain, and phosphorylation site is depicted as a “P” in the black circle.