Proteins in the ovular secretions of conifers




O'Leary, Stephen James Bernard

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Most conifers employ a liquid secretion originating fiom within the ovule at some point during reproduction. Although widely known, these ovular secretions have been poorly characterized. Biochemical analyses of these liquids have been limited to reports of sugars, amino acids, organic acids, and calcium. The purpose of this study was to investigate the physiological regulation of conifer ovular secretions and to further elucidate their contents. Postpollination droplet production was measured in three hybrid larch trees (Larix x marschlinsii Coaz) in relation to xylem water tension in the stem. Secretion production was not correlated to the predictable diurnal fluctuation of tree water status. The ovular secretions of this species were found to be independent of the physiological condition of the stem and are likely under the control of local structures such as the cones or ovules. The concentrations of glucose, fi-uctose, and sucrose were measured in the secretions of larch and hybrid yew (Taxus x media Rehder). In agreement with results &om other conifers, the concentrations of glucose (156 mM) and fructose (145 mM) in the larch secretion were found to be higher than sucrose (1 08 mM). The pollination droplet of yew displayed a novel pattern. The sucrose concentration in this species (23 mM) was found to be an order of magnitude higher than either glucose (2.7 mM) or hctose (2.1 mM). The ovular secretions of larch, yew, Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), and western red cedar (Thuja plicata Donn. ex D. Donn) were found to contain complex mixtures of proteins when examined by polyacrylamide gel electrophoresis or reversed phase high performance liquid chromatography. The proteins of larch and yew were produced consistently from tree to tree and throughout the period of secretion production. N-terminal amino acid sequencing and antibody recognition identified proteins in larch and yew samples believed to be involved in pollen germination and the promotion of pollen tube elongation. The cell wall modifLing enzyme xyloglucan endotransglycosylase (XET) was identified in the larch secretion. Immunolocalization identified cells in the apical region of the larch micropyle as the site of XET production. Arabinogalactan proteins (AGPs), known to promote pollen tube growth in angiosperms, were found in the secretions of both conifer species. AGP production in the yew ovule was localized to the nucellus. Four pathogenesis-related (PR) proteins were identified in the larch and yew ovular secretions. A lipid transfer protein (LTP) belonging to the PR-14 group was identified in the larch secretion by N-terminal amino acid sequencing. A thaumatin-like protein (TLP, PR-5) was tentatively identified in the larch sample by antibody recognition. One acidic and one basic TLP were identified in the yew secretion by tandem mass spectrometry (MSMS) sequencing of internal peptide fragments. These proteins were named TmTLPa and TxmTLPb respectively. MSMS sequencing also identified a P-1,3-glucanase of the PR-2 group in the yew secretion (TmpGlu). The cDNA coding for TxmTLPa was sequenced and assessed for heterologous protein expression. The nucleic acid sequence predicts a preprotein of 233 amino acid residues with a 28 residue export signal. The putative mature protein has a predicted molecular weight of 21.40 kDa and pI of 4.4. The deduced protein sequence contains 16 cysteine residues conserved across TLPs, and five residues that contribute to the acidic cleft of antifbngal TLPs. In order to produce Z'xmTLPa in sufficient quantities to perform bioassays, the mature sequence of this protein has been inserted into a plasmid vector for the expression of a TxmTLPa fusion protein. This report contains the first simultaneous study of ovular secretion production and tree water status, the first measurements of the sugar concentrations in the ovular liquids of L. x marschlinsii and T. x media, and the first identification of proteins in the ovular secretion of any seed plant.