Cysteine Rich Proteins and pollen-stigmatic compatibility in Arabidopsis

Abstract

During sexual reproduction in angiosperms, successful pollination relies on a complex series of interactions between cells of male and female reproductive structures and gametes. The first stage of this process amongst members of the Brassicaceae involves a series of interactions between pollen grains and stigmatic papilla cells, where compatible pollen is recognised by the papilla cell and permitted to hydrate and germinate the pollen tube, which will grow into the style and reach the ovule to release the sperm, result in fertilization. It has been proposed that a “basal compatibility” signalling system is crucial for the establishment of compatibility between the pollen and stigmatic surface upon the first few minutes of pollen arrival. Previous research in Doughty lab based on proteomic analyses of Arabidopsis thaliana and Brassica pollen coat has led to the identification of a diverse grouping of pollen coat-borne small cysteine-rich proteins (CRPs) of unknown functions; These small CRPs, now known as pollen coat proteins (PCPs), are considered potential regulators of pollen-stigma
compatibility. In this project a wide range of PCPs has been identified from the pollen coat proteome of Arabidopsis thaliana and a classification of PCPs has been made based on sequential and gene ontology analysis. Amongst the six major families of PCPs identified, one group of PCPs namely PCP-Bs has raised our interest due to its putative role in regulating the pollen-stigma compatibility at the early stage of pollination. A new member of PCP-B family, namely PCP-Bε, has been identified in this research. By taking mutational approach, a collection of single and combined pcp-b gene knockout mutants has been generated, in vivo bioassays revealed that pcp-bε single and combined mutant lines possess a more severe pollen hydration defect than that of the other pcp-b mutants, suggesting that PCP-Bε protein plays a more essential role in the establishment of pollen-stigma compatibility. The phylogenetic analysis and molecular evolutionary study of genes encoding PCP-Bs and PCP-B-like proteins has revealed birth-and-death
evolution of PCP-B related genes throughout the evolutionary history of Brassicaceae species. Furthermore, population genetics has shown members of AtPCP-Bs possess different evolutionary characters, suggesting functional diversification of the AtPCPBs. To gain more understanding of the putative stigmatic targets of PCPs that found to be influencing compatibility, heterologous expressed AtPCP-Bs were used as baits in protein-protein interaction (Oliveira et al.) assays to identify their potential receptors in isolated stigmatic proteins. This project shed more light on the roles that played by small pollen coat CRPs in plant reproductive signalling and provided valuable insight on the biological and evolutionary significance of AtPCP-B family. More widely, this research could lead to a better understanding of what regulates compatibility at the interspecific level, which has important implications for plant breeding programmes and could eventually lead to the development of new tools to facilitate hybridization in crop
improvement.

Date of Award	4 Nov 2020
Original language	English
Awarding Institution	University of Bath
Supervisor	James Doughty (Supervisor) & Roderick Scott (Supervisor)