Revealing phosphoproteins playing role in tobacco pollen activated in vitro

Abstract

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.

Cytological and Proteomic Analyses of Osmunda cinnamomea Germinating Spores Reveal Characteristics of Fern Spore Germination and Rhizoid Tip Growth*

“…However, the fern spore germination happens in the medium, therefore, it is somewhat more complex and affected by various environmental factors, such as light, gravity, calcium, phytohormone, and temperature (6). The protein functional skew in fern spores and pollen grains revealed from this study and recent proteomics studies on germinating pollen from kiwifruit (Actinidia deliciosa) (71,72), Nicotiana tabacum (73), rice (Oryza sativa) (26), Brassica napus (74), Pinus strobus (75), Picea meyeri (76,77), Picea wilsonii (78) and Arabidopsis (79,80) presented new evidence for the differentiation of fern spores and spermatophyte pollen (Fig. 10).…”

“…No photosynthetic proteins were found in pollen. This highlights that the remodeling of pollen tube cell wall, the loosening and hydrolyzing stigma surface cell and transmitting track of pistil, The data of germinating pollen were summarized and reclassified based on pollen proteomic studies on Oryza sativa (26), Actinidia deliciosa (71,72), Nicotiana tabacum (73), Brassica napus (74), Pinus strobus (75), Picea meyeri (76,77), Picea wilsonii (78), and Arabidopsis thaliana (79,80). The y axis represents the reclassified functional categories, and the x axis shows the percentage of identified proteins.…”

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro

“…These 432 unique phosphorylation sites have been assigned to 301 individual proteins. The number of phosphorylation sites identified represents a great improvement in comparison to our previous tobacco male gametophyte phosphoproteomic study that identified 52 unambiguous phosphorylation sites (21). In that study we applied Al(OH) 3 -metal oxide/hydroxide affinity chromatography for phosphoprotein enrichment (allowing the annotation of only one phosphorylation site), and TiO 2 phosphopeptide enrichment for the analysis of phosphorylation sites of selected candidate proteins from mature pollen.…”

“…Seven synthetic peptides were spiked to the peptide mixture in order to check the reproducibility of the replicates. The phosphopeptides bound to TiO 2 beads were washed and eluted as described previously (21,29).…”

“…In this study, Ischebeck and colleagues compared the proteome of eight male gametophyte stages ranging from diploid microsporocytes to pollen tubes. Interestingly, the first tobacco pollen phosphoproteomic paper appeared earlier than the whole proteome was published (21). In order to identify phosphoproteins in tobacco mature pollen and pollen activated in vitro for 30 min, metal oxide/hydroxide affinity chromatography phosphoprotein enrichment employing an aluminum hydroxide matrix (Al(OH) 3 ) was carried out (22).…”