Application of proteomics to the study of pollination drops

Abstract

Premise of the study: Pollination drops are a formative component in gymnosperm pollen-ovule interactions. Proteomics offers a direct method for the discovery of proteins associated with this early stage of sexual reproduction. •

Methods: Pollination drops were sampled from eight gymnosperm species: Chamaecyparis lawsoniana (Port Orford cedar), Ephedra monosperma, Ginkgo biloba, Juniperus oxycedrus (prickly juniper), Larix ×marschlinsii, Pseudotsuga menziesii (Douglas-fir), Taxus ×media, and Welwitschia mirabilis. Drops were collected by micropipette using techniques focused on preventing sample contamination. Drop proteins were separated using both gel and gel-free methods. Tandem mass spectrometric methods were used including a triple quadrupole and an Orbitrap. •

Results: Proteins are present in all pollination drops. Consistency in the protein complement over time was shown in L. ×marschlinsii. Representative mass spectra from W. mirabilis chitinase peptide and E. monosperma serine carboxypeptidase peptide demonstrated high quality results. We provide a summary of gymnosperm pollination drop proteins that have been discovered to date via proteomics. •

Discussion: Using proteomic methods, a dozen classes of proteins have been identified to date. Proteomics presents a way forward in deepening our understanding of the biological function of pollination drops.

Keywords: conifers; gnetophytes; gymnosperm; mass spectrometry; pollination drop; proteomics.

Fig. 1.

Pollination drop collection. (A) Pollination drop of Ginkgo biloba, 20×. (B) RNA-ase free micropipette tip with filter. (C) Drops are aggregated into a 2-mL microtube by blowing out the pipette tip.

Fig. 2.

One-dimensional SDS-PAGE of various conifer pollination drop proteins. The gel was stained using GelCode Blue. Fifty microliters of sample was loaded onto a precast 4–12% Invitrogen gel and run for 1 h at 4°C. The gel was run at 118 mA through the stacking gel and 70 mA through the separating gel. Lanes: (1, 7) molecular weight ladder, (2) Pseudotsuga menziesii (Douglas-fir), (3) Larix ×marschlinsii (hybrid larch), (4) Taxus ×media (hybrid yew), (5) Chamaecyparis lawsoniana (Port Orford cedar), (6) Juniperus oxycedrus (prickly juniper).

Fig. 3.

Reversed-phase profile and spectra of three gymnosperm taxa. (A) RP-HPLC profiles of two Larix ×marschlinsii ovular secretion samples. One sample was collected at the beginning of the secretion period (red trace) and the other collected seven days later (black trace). In each experiment, 20 μL of whole sample was loaded onto a C8 column and separation occurred in a linear gradient of increasing acetonitrile concentration. UV absorbance of eluent was monitored at 220 nm. Asterisks denote fractions shown by SDS-PAGE to contain protein. (B) MS/MS fragmentation data. Tryptic peptide from chitinase protein found in Welwitschia mirabilis pollination drops introduced by nanospray electrospray ionization into a QSTAR Pulsar I Hybrid Quadrupole-TOF MS/MS mass spectrometer (Applied Biosystems/MDS Sciex). Data were managed with PEAKS (Bioinformatics Solutions) and Bioanalyst software (Applied Biosystems/MDS Sciex). (C) MS/MS fragmentation data. Peptide (VYSGDTDGRVP) from serine carboxypeptidase II-3 protein found in Ephedra monosperma pollination drops introduced by nanospray electrospray ionization into the LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Data were managed with Proteome Discoverer (Thermo Fisher Scientific) and Mascot version 2.2.1 (Matrix Science) software.

Fig. 4.

Flow chart of proteomics protocol for pollination drops.