ORIGINAL ARTICLE Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions Rebecca E. Wagner Æ Serena Mugnaini Æ Richard Sniezko Æ Darryl Hardie Æ Brett Poulis Æ Massimo Nepi Æ Ettore Pacini Æ Patrick von Aderkas Received: 20 April 2007 / Accepted: 25 August 2007 / Published online: 16 October 2007 Ó Springer-Verlag 2007 Abstract The pollination droplet is a highly conservative pollination mechanism that is observed in all major gym- nosperm taxa. Proteomics analysis of the pollination drops was carried out on four gymnosperm species: Juniperus communis (common juniper), Juniperus oxycedrus (prickly juniper), Chamaecyparis lawsoniana (Port Orford cedar), and Welwitschia mirabilis. Pollination drop proteins were purified by SDS-PAGE, and the most abundant proteins were analyzed by mass spectrometry and sequenced. Based on BLAST searching of combined amino acid sequences, the following proteins were identified in the following species: an 83-kDa subtilisin-like proteinase, a 62-kDa glycosyl hydrolase, a 47.5-kDa glucan 1,3-b-glucosidase precursor, a 30-kDa chitinase, and a 25-kDa thaumatin-like protein were identified in J. communis; a 30-kDa chitinase, a 25-kDa thaumatin-like protein, and a 32.5-kDa glucanase-like pro- tein were identified in J. oxycedrus; an 83-kDa subtilisin-like proteinase, a 62-kDa b-D-glucan exohydrolase, a 47.5-kDa glucan 1,3-b-glucosidase, and two 25-kDa thaumatin-like proteins were identified in C. lawsoniana, and a 25-kDa chitinase was identified in W. mirabilis. Based on protein identifications, there is strong evidence that the pollination drop functions in both pathogen defense and pollen devel- opment. The discovery of similarities in terms of peptide sequence and protein identifications indicates that ovular secretions are functionally conservative, and that they are essential to reproductive success. Keywords Gymnosperm reproduction Á Pollination drop Á Proteomics Á Juniperus Á Chamaecyparis Á Welwitschia Introduction Virtually all gymnosperms depend on wind to deliver pollen from male to female cones. The pollen capture strategy employed by the female cone is highly evolved. Pollination mechanisms include the presence of sticky hairs, the engulfment of pollen by asymmetric ovular growth, extended siphonogamy, and secretions from the ovule (Gelbart and von Aderkas 2002). Pollination drops exude from the ovule, capture pollen directly from the air, and retract into the ovule, carrying the pollen with them. Thus, these secretions trap and transport pollen within the ovule. Ovular secretions are present in all major extant and extinct gymnosperm taxa and are crucial to seed plant reproduction. The study of ovular secretion composition began in the second half of the twentieth century (McWilliam 1958; Ziegler 1959), yet the first in-depth studies only appeared in more recent years (Owens and Blake 1984; Owens et al. 1987; Se ´ridi-Benkaddour and Chesnoy 1988; Carafa et al. 1992; Xing et al. 2000). Early studies reported relatively simple water-soluble compounds Communicated by Yongbiao Xue. R. E. Wagner Á B. Poulis Á P. von Aderkas (&) Graduate Centre for Forest Biology, Department of Biology, University of Victoria, V8W 3N5 Victoria, BC, Canada e-mail: pvonader@uvic.ca S. Mugnaini Á M. Nepi Á E. Pacini Department of Environmental Sciences ‘‘G. Sarfatti’’, University of Siena, Via P.A. Mattioli 4, 53100 Siena, Italy R. Sniezko USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, OR 97424, USA D. Hardie University of Victoria, Genome BC Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St, V8Z 7X8 Victoria, BC, Canada 123 Sex Plant Reprod (2007) 20:181–189 DOI 10.1007/s00497-007-0054-8