BIOLOGIA PLANTARUM 55 (3): 499-506, 2011 499 Impact of homogenization and protein extraction conditions on the obtained tobacco pollen proteomic patterns J. FÍLA 1,2 , V. ČAPKOVÁ 1 , J. FECIKOVÁ 1 and D. HONYS 1 * Laboratory of Pollen Biology, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, CZ-16502 Prague, Czech Republic 1 Department of Plant Experimental Biology, Faculty of Science, Charles University in Prague, Viničná 5, CZ-12844 Prague, Czech Republic 2 Abstract Mature pollen grain represents a highly desiccated structure with an extremely tough cell wall. Thanks to it, it resists common proteomic protocols. Instead, a robust homogenization has to be performed since proteins are needed to burst out of the cell to be included in the extracted proteome fraction. Here, a novel way of pollen homogenization employing Roche MagNA Lyser Instrument is presented, sparing time and laborious work. However, plant proteomics does not rely solely on perfect homogenization; also the choice of the extraction protocol is of key importance. The composition of the extraction buffer has a decisive influence on which proteome fraction will be extracted. Therefore the second part of our study is dedicated to the comparison of different extraction protocols with respect to subsequent proteomic analyses. Additional key words: proteomics, Roche MagNA Lyser Instrument. Introduction Unlike animals, plants consist of cells enclosed in resistant cell walls and often loaded with a variety of interfering compounds, e.g. phenolic compounds, alkaloids and other secondary metabolites. Moreover, pollen grains have an even harder cell wall and are fully desiccated to withstand many environmental stresses and to protect the genetic information as well as stored compounds needed for all post-pollination processes including massive pollen tube growth leading to the subsequent successful fertilization. Pollen represents a very interesting model for the studies of cell polarity (Hepler et al. 2001) and tip growth (Geitmann and Steer 2006). During germination, pollen grain rehydrates and develops into a metabolically extremely active, rapidly growing pollen tube. To achieve such an explosive growth, pollen grain has to store large amounts of RNA and protein reserves (Tupý 1982, Honys and Čapková 2000, Honys et al. 2000). During pollen maturation, both transcription and translation play an important role in the global and specific gene expression patterns. On the contrary, the germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation (Twell 1994). In tobacco, transcription was detected only during the first several hours of the pollen tube growth, which led to the conclusion that pollen tube growth is mainly dependent on translation (Čapková et al. 1988). A significant fraction of mRNAs is stored in the form of translationally-inactive EDTA/puromycin-resistant RNP complexes (EPPs; Honys et al. 2000, 2009). Proteome does not exactly correspond to the transcriptome (De Groot et al. 2007). Also in Arabidopsis thaliana pollen, mRNAs of some abundant proteins are present at very low concentrations or even not present at all and vice versa, mRNA abundance can be higher than that of corresponding proteins (Holmes-Davis et al. ⎯⎯⎯⎯ Received 29 November 2009, accepted 9 April 2010. Abbreviations: BSA - bovine serum albumine; CHAPS - 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; DTT - dithiothreitol; IEF - isoelectric focusing; PMSF - phenylmethanesulphonyl fluoride; SB 3-12 - sulfobetaine-12; SDS-PAGE - sodium-dodecyl-sulfate polyacrylamide gel electrophoresis; TCA - trichloroacetic acid. Acknowledgements: This work was supported by ESF (COST Action FA 0603), the Ministry of Education of the Czech Republic (MSMT grant No. OC08011) and Czech Science Foundation (grant No. P501/11/1462). * Corresponding author; fax: (+420) 225106456, e-mail: honys@ueb.cas.cz