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...

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