Isolation and characterization of pollen coat proteins of Brassica campestris that interact with S locus-related glycoprotein 1 involved in pollen-stigma adhesion

Adhesion of pollen grains to the stigmatic surface is a critical step durin g sexual reproduction in plants. In Brassica, S locus-related glycoprotein 1 (SLR1), a stigma-specific protein belonging to the S gene family of prote ins, has been shown to be involved in this step. However, the identity of t he interacting counterpart in pollen and the molecular mechanism of this in teraction have not been determined. Using an optical biosensor immobilized with S gene family proteins, we detected strong SLR1-binding activity in po llen coat extracts of Brassica campestris. Two SLR1-binding proteins, named SLR1-BP1 and SLR1-BP2, were identified and purified by the combination of SLR1 affinity column chromatography and reverse-phase HPLC, Sequence analys es revealed that these two proteins (i) differ only in that a proline resid ue near the N terminus is hydroxylated in SLR1-BP1 but not in SLR1-BP2, and (ii) are members of the class A pollen coat protein (PCP) family, which in cludes PCP-A1, an SLG (S locus glycoprotein)-binding protein isolated from Brassica oleracea. Kinetic analysis showed that SLR1-BP1 and SLR1-BP2 speci fically bound SLR1 with high affinity (K-d = 5.6 and 4.4 nM, respectively). The SLR1-BP gene was specifically expressed in pollen at late stages of de velopment, and its sequence is highly conserved in Brassica species with th e A genome.