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. Molecular & Cellular

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

“…The finding that we could discriminate between pollen populations with and without germination capacity at 3 and 12 MHz demonstrates that we were able to detect the described metabolic changes pollen germination [19,26–28,41]. Pollen incapable of active re-hydration has a lower water content and likely a slower (passive) up-take than hydrated pollen and subsequently a different resistance when exposed to an electric field, which allows the discrimination between the two populations by IFC and leads to a high predictability of the IFC data for in vitro germination as only hydrated pollen is able to germinate [26,27,41].…”

“…The dielectric principles of IFC predict an optimal frequency range for cell size analysis between 0.01 and 20 MHz, membrane integrity between 1 and 10 MHz, and cytoplasmic conductivity above 10 MHz [2]. Pollen becomes metabolic active prior germination, thus changes in membrane integrity and cytoplasmic conductivity should be detectable when comparing germinating active with inactive pollen [28]. …”

“…The ability of tomato pollen to germinate after long-term storage or heat stress depends on the biosynthesis of polyamines [60,61], or β -alanine [62] with a role for specific amino acid transporters during de-/hydration phases [63]. In tobacco ( Nicotiana tabacum ) pollen specific peptides are dephosphorylated during pollen activation [28]. …”

“…During this phase the metabolic activity of the pollen grain increases, which is essential for the later pollen tube formation [28]. Pollen tube elongation is controlled by active vesicle and organelle trafficking along an intact cytoskeleton [29].…”

Proteomes and Phosphoproteomes of Anther and Pollen: Availability and Progress

“…Therefore, systematical and comprehensive identification of phosphoproteins in anthers should be very useful for exploring the signaling pathways that mediate anther development and give insight into pathways and protein interaction networks underlying anther development. At present, six anther related phosphoproteomic analysis were reported in angiosperm (Table ), which provide useful information about protein phosphorylation and its regulation during anther development. Anther organ contain mixtures of different cell types which decreases the resolution and selectivity of specific phosphoproteins in specific cell types.…”