Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth

Abstract

Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro

“…Out of these 837 proteins, 120 fell into the GO category "not assigned", that represented approx. 14% of all pollen-specific proteins reported by Ischebeck and colleagues (20). From this point of view, our phosphoproteomic data set is consistent with the published tobacco male gametophyte proteome.…”

“…Interestingly, most of our zinc finger transcription factors showed also prolyl-directed phosphorylation motif making them likely substrates of MAP kinases (60). Some MAP kinases were already identified in tobacco male gametophyte (20,61,62). However, experimental data directly linking these MAP kinases to their targets have yet to be established.…”

“…In tobacco proteome, it was clearly shown that gametophytic tissues contained specific proteins (837 out of 2135 proteins) that were not shared by sporophyte tissues, particularly leaves and roots (or were at least not as abundant as in gametophyte, and so remained below the detection limit of the proteomic techniques; ref. 20). Out of these 837 proteins, 120 fell into the GO category "not assigned", that represented approx.…”

“…60). Both these large kinase families were identified in the tobacco male gametophyte proteome (20), supplemental Table S5. MAPKs play a key regulatory role in many physiological processes including stress reactions and pollen hydration (62).…”

“…The tobacco pollen proteome was studied directly by a high-throughput approach but appeared only recently (20). In this study, Ischebeck and colleagues compared the proteome of eight male gametophyte stages ranging from diploid microsporocytes to pollen tubes.…”