CLONING OF PCP1, A MEMBER OF A FAMILY OF POLLEN COAT PROTEIN (PCP) GENES FROM BRASSICA-OLERACEA ENCODING NOVEL CYSTEINE-RICH PROTEINS INVOLVED IN POLLEN-STIGMA INTERACTIONS

The pollen coatings of both Brassica oleracea and Brassica napus conta in a small family of basic 6-8 kDa proteins which are released on to t he stigmatic surface on pollination. Following partial amino-acid sequ encing of one of these pollen coat proteins (PCPs), PCR primers were c onstructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, reve aled a characteristic pattern of expression during late stages of anth er development. Interestingly, in situ hybridization revealed expressi on of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybrid ization experiments have shown the gene (PCP?) to be a member of a lar ge family of between 30 and 40 PCP genes in the genome of Brassica ole racea. Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F-2 segregants, perhaps resulting from t he clustering of PCP sequences. PCP1 contains a single intron and enco des a small, basic peptide 83 amino acids in length featuring a hydrop hobic signal peptide sequence separated from the more hydrophilic, cys teine-rich mature protein. The central part and C-terminal region of t he peptide contain a characteristic and invariant pattern of eight cys teines which show clear homology with a number of other anther-specifi c genes; the remainder of the sequence shows little similarity to othe r sequences on the data bases. The product of PCP? is a member of a la rge family of similar proteins, some of which have been demonstrated t o bind specifically to S-locus glycoproteins, but does not appear to b e genetically linked to the S-locus.