Mass spectrometry-based proteomics techniques are now proving to be useful for evolutionary plant biologists. What was originally thought to be a receiver of pollen,1 pollen drops are now known to contain a specialized assortment of proteins. To further investigate changes in protein levels, Prior et al.2 performed a proteomic analysis of pollen drops found in gymnosperms.
Pollination drops from eight gymnosperm species were obtained using micropipettes and analyzed using four proteomics methods. Based on previous understanding of protein complexities, it was necessary to use a varied approach for different species. The majority of samples were separated using gel electrophoresis, unless they were known to be viscous, sugar-rich, and debris-laden pollination drops, such as those from W. mirabilis
L. ×marschlinsii samples were separated by a combination of RP-HPLC and gel electrophoresis Following separation based on the work of Wagner et al.,3 proteins were then reduced, alkylated, and digested with trypsin prior to SDS-PAGE.
W. mirabilis and J. oxycedrus samples were run on a QSTAR Pulsar I hybrid quadrupole-TOF MS/MS mass spectrometer (Applied Biosystems). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using a QTRAP hybrid triple quadrupole/linear ion trap MS/MS mass spectrometer equipped with a nano-electrospray ionization source (Applied Biosystems/MDS Sciex) coupled to an integrated Famos autosampler, Switchos switching pump, and UltiMate Micro Pump system (LC Packings).
Ephedra monosperma samples were separated by online reversed-phase chromatography using a Thermo Scientific EASY-nLC II system and identified using an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). The raw data files were searched using Proteome Discoverer software version 1.2 (Thermo Scientific) with the Mascot version 2.2.1 search engine, UniProt-SwissProt 20110104 (523,151 sequences; 184,678,199 residues), and Viridiplantae database. All sequences were manually verified.
Results revealed species-specific differences, with closely related conifers more similar in composition than more distantly related conifers. Proteins collected within a species at different times were comparable. One problem Prior et al.2 ran into was a lack of hits in the database. Only half of proteins discovered in Juniperus could be identified, which is evidence of further work that needs to be done. “The proteins we are finding are really starting points for other research,” says Prior. She hopes this research will lead to further discoveries in the evolution of gymnosperms.4
1. Singh, H. (1978) Embryology of Gymnosperms, Stuttgart, Germany: Gebrüder Borntraeger
2. Prior, N., et al. (2013) ‘Application of proteomics to the study of pollination drops‘, Applications in Plant Sciences, 1 (4), (p. 1300008)
3. Wagner, R., et al. (2007) ‘Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions‘, Sexual Plant Reproduction, 20 (4), (pp. 181-189)
4. American Journal of Botany (2013) ‘Metabolic fingerprinting: using proteomics to identify proteins in gymnosperm pollination drops‘, ScienceDaily, Retrieved April 29, 2013