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.
Highlights
Plants contain numerous specialized cell types, each of them expressing a specific set of proteins
Isolation and Proteomic Analysis of Early and Late Pollen Developmental Stages—Pollen from a total of eight developmental stages was harvested for proteomic analysis (Figs. 1 and 2)
Pollen from the microsporocyte and meiosis stages was obtained as large aggregates, which were associated with cell debris (Fig. 1), it was possible to isolate individual cells from later stages (Figs. 1 and 2)
Summary
Plant Growth and Pollen Collection—Tobacco was grown under greenhouse conditions (12 h of light, 120 mol mϪ2 sϪ1, 23 °C during the day, 20 °C at night, 60% humidity). The pollen fragments were resuspended in 200 l of protein extraction buffer (62.5 mM Tris-HCl pH 6.5, 5% SDS (w/v), 10% glycerol (v/v), 10 mM DTT, 1.2% (v/v) plant protease inhibitor mixture (Sigma P9599)) and incubated for 5 min at room temperature After this time, the samples were mixed again by pipetting, incubated for 3 min at 90 °C, and centrifuged at 21,000 ϫ g for 5 min at room temperature. All proteins in the three used databases were blasted for the closest Arabidopsis (TAIR10) homologue using an unpublished Python script in conjunction with stand-alone BLAST v2.2.26ϩ using the default matrix, and entries in the TAIR Arabidopsis MapMan mapping file (Ath_AGI_LOCUS_TAIR10_Aug2012) were replaced as previously described [54]. Further blasting of the tobacco protein sequences versus the list of Arabidopsis proteins found in Arabidopsis pollen [14] and a list of pollen-affected Arabidopsis mutants (extended list from Ref. 14) was performed using the same Python script
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