| Summary: | The early stages of post-pollination in angiosperms involve multiple phases of interaction between male and female reproductive tissues. The establishment of the pollen-stigma interaction is proposed to involve a basal compatibility system that enables compatible pollen to be recognised by the receptive stigma. Divergence of components involved in this system could facilitate the establishment of prezygotic breeding barriers that would limit wasted mating opportunities, restrict interspecies gene flow and contribute to reproductive isolation. A diverse family of small secreted cysteine-rich proteins (CRPs) found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important regulators of the pollen-stigma interaction. One class of PCPs isolated from the pollen coat of Brassica oleracea, the PCP-Bs, have previously been described, but their function was unknown. In this study, four putative Arabidopsis thaliana PCP-B-encoding genes were identified, determined to be gametophytically expressed during the late stages of pollen development and confirmed as pollen coat proteins. Bioassays utilising single and multiple pcp-b gene knockouts revealed that AtPCP-Bs function in the early stages of post-pollination. To identify the stigmatic targets of AtPCP-B ligands, a series of protein-protein interaction (PPI) assays were carried out with heterologously expressed AtPCP-Bs and isolated stigmatic proteins. To provide insight into the evolutionary characters of PCP-Bs, phylogenetic analysis and molecular evolutionary study revealed evidence of positive selection acting on sites of genes encoding PCP-Bs and PCP-B-like proteins. Such evidence suggests that AtPCP-Bs are important components of the basal compatibility system by establishing a molecular dialogue between compatible pollen grains and the stigma. Proteomic analyses of pollen coat from Arabidopsis thaliana and Brassica oleracea uncovered large numbers of small CRPs, which may act as important regulatory factors of pollen-stigma interaction. This project shed new light on the biological and evolutionary significance of pollen coat CRPs in plant reproductive signalling.
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