Pollen and Pollen Tube Biology

0541115 - ÚEB 2021 RIV US eng M - Monography Chapter
Hafidh, Said - Honys, David
Isolation of the pistil-stimulated pollen tube secretome.
Pollen and Pollen Tube Biology. Vol. Methods in Molecular Biology. New York: Humana Press, 2020 - (Geitmann, A.), s. 41-72. ISBN 978-1-0716-0671-1
R&D Projects: GA MŠMT(CZ) LM2018129; GA ČR(CZ) GA17-23203S; GA ČR(CZ) GA18-02448S; GA ČR(CZ) GA18-07027S; GA MŠMT(CZ) EF16_019/0000738
Grant - others:OPPK(XE) CZ.2.16/3.1.00/21519
Institutional support: RVO:61389030
Keywords : Cell–cell communication * Glycosylation * lc-ms/ms * Pollen tube guidance * Protein secretion * Sexual plant reproduction
OECD category: Biochemical research methods
http://doi.org/10.1007/978-1-0716-0672-8_4

Detection of secreted proteins and peptides during pollen tube guidance has been impeded due to lack of techniques to capture the pollen tube secretome without contamination from the female secreted proteins. Here we present a protocol to detect tobacco pollen tube secreted proteins, semi-in vivo pollen tube secretome assay (SIV-PS), following pollen tube crosstalk with the female reproductive tissues. This method combines the advantages of in vivo pollen tube–pistil interaction and filter-aided sample preparation (FASP) techniques to obtain an in-depth proteome coverage. The SIV-PS method is rapid, efficient, inexpensive, does not require specialized equipment or expertise, and provides a snapshot of the ongoing molecular interplay. We show that the secretome obtained is of greater purity (<,1.4% ADH activities) and that pollen tubes are physiologically and cytologically unaffected. A compendium of quality controls is described and a rough guide on downstream bioinformatics analysis is outlined. The SIV-PS method is applicable to all studies of protein secretion using pollen tube as a model and can be easily adapted to other flowering species with modification. The overall duration for this protocol is approximately 8 hours spanning 4 days (an average of 2 h/day per two workers) excluding microscopy and LC-MS/MS analysis.
Permanent Link: http://hdl.handle.net/11104/0318709