Interaction between a coating-borne peptide of the Brassica pollen grain and stigmatic S (self-incompatibility)-locus-specific glycoproteins.

Abstract

Methods are described for the removal of the sporophytic pollen grain coating of Brassica olracea and for the isolation of coat polypeptides. The coat contains a small number of proteins ranging from 6 to 45 kDa. Many of the larger proteins are glycosylated, while all carry hig positive charges resulting in pI values from 8.5 to 11. Polypeptides with pI values of 9.5, 9.0, and 8.5 possess strong esterase activity. No major differences could be detected in either pI values or molecular masses of pollen-coating polypeptides from grains carrying different sporophytically expressed S (self-incompatibility) alleles. Mixing pollen coat proteins with smatic extracts results in a conspicuous binding interaction involving female S-locus-specific and perhaps S-locus-related glycoproteins. This interaction, which is reversed by heating in the presence of SDS, results in an apparent charge shift of the female glycoprotein(s) of up to 2 pI units. The male participant in this interaction has been isolated by using a combination of fast protein liquid chromatography and reverse-phase HPLC and was shown to be a 7-kDa nonglycosylated peptide. Experiments with whole pollen cultured in vitro show challenge with stigmatic extracts to stimulate the release of gametophytic and sporophytic polypeptides and to result in the formation of a conspicuous interaction product, demonstrating the 7-kDa peptide to be freely available within the coating of pollen in vivo.The self-incompatibility (SI) systems of higher plants are emerging as a family of unique signaling systems evolved from mechanisms already operative in the pollen/pistil interaction (1). Thus, in Nicotiana and other members of the Solanaceae, self-pollen is rejected by a mechanism involving a stylar RNase (2, 3), an enzyme common to the styles of many plants and hypothesized to play a role in defense against pathogens. In most species, SI is regulated by a simple genetic system based on few loci and large numbers of alleles (4, 5); generally, if pollen and stigma carry alleles in common, the pollen/stigma interaction is disrupted. The genetics of SI in Brassica and other members of the Cruciferae and Compositae is not so straightforward; the pollen S phenotype is determined by the S-allelic constitution of the parent plant, rather than that of the haploid grain (6).Through a series of elegant molecular studies, Nasrallah et al. (7) have demonstrated two types of sequence to be linked to the S locus of Brassica. One (S locus glycoprotein; SLG) encodes a 55-kDa glycoprotein (S-locus-specific glycoprotein; SLSG) expressed in the stigmatic papillae, and the other encodes a transmembrane kinase (S receptor kinase; SRK) expressed in the organs ofboth sexes. Sequence comparisons suggest that the SRK features a domain, which may be extracellular, with maintained homology to the SLG. Further, other gene families with considerable homology to the SLG are also present in the genome (8-10) but unlinked to the S locus (S locus related; SLR). Interestingly, reporter constructs driven...

Characterization of anther‐expressed genes encoding a major class of extracellular oleosin‐like proteins in the pollen coat of Brassicaceae†

“…Rapid washes of mature Brassica pollen grains have previously been shown to result in the complete removal of the tryphine with no apparent damage to either the extracted polypeptides or the remaining coatless pollen (Doughty et al, 1993). This was confirmed in the present study where cyclohexane washes of mature B. napus cv.…”

Pollen and Stigma Structure and Function: The Role of Diversity in Pollination

“…These cells secrete oil droplets and proteins and subsequently degenerate, filling the cavities of the exine with their cytoplasmic contents. Compared with sporopollenin-rich exines, pollen coats are better characterized; they can be extracted and their contents can be purified in sufficient quantity for identification (Doughty et al, 1993;Ross and Murphy, 1996;Ruiter et al, 1997;Murphy and Ross, 1998;Mayfield et al, 2001). The first biochemical isolations were in Liliacea, in which pollen coats were found to be largely lipidic, with carotenoids as major pigments (Heslop-Harrison, 1968).…”

“…In Arabidopsis, the pollen coat contains longand short-chain lipids along with a small set of proteins, including six lipases and eight Gly-rich oleosin proteins that contain a lipid binding domain (Mayfield et al, 2001;Fiebig et al, 2004). The pollen coating of Brassica is structured similarly and also has been shown to contain peptides involved in self-incompatibility (Doughty et al, 1993). Disrupting pollen coat lipids or pollen coat proteins in Brassicaceae species can delay or block pollen hydration.…”

Characterization of Oleosins in the Pollen Coat of Brassica oleracea

“…Safe carriage of this information from the anther to a receptive stigma Is ascertained by a thick wall around the pollen grain. In many species, the outermost layer of this wall is a lipidic coating, containing proteins and carbohydrates, that is well suited to protect the poilen pro toplast and to mediate pollen transport, but it is also equipped to play a role in pollen-stigma interactions (HesiopHarrison, 1979;Knox, 1984;Elleman and Dickinson, 1990;Elleman et a!., 1992;Dickinson, 1993;Doughty etal., 1993).…”