Pollination in lily: Discovery of a chemotropic molecule in the stigma

Pollination in flowering plants is the process of delivering two sperm cells, through the stigma, style and ovary, to the ovule for double fertilization. Pollen tube guidance by female tissues in the pistil is essential for successful fertilization. In lily, pollen tubes grow by interacting with specialized extracellular matrices of the epidermis through the open stigma and style. In this dissertation, new pollen tube guidance molecules from the lily stigma and style are discovered.; In Chapter 1, the first chemotropic peptide described for plants is purified biochemically. Further study using proteomics and molecular biology showed that the peptide is a plantacyanin, a member of the blue copper protein or phytocyanin family. The plantacyanins (basic blue proteins) are cell wall proteins of unknown function. We have named the lily plantacyanin, chemocyanin. Pollen tube chemotropism in the presence of chemocyanin is enhanced by stigma/stylar cysteine-rich adhesin (SCA). SCA is a previously identified pollen tube adhesion molecule isolated from the lily style.; In Chapter 2, immunolocalization studies were conducted to elucidate the function of SCA in the stigma and the style at the light microscopic and at the transmission electron microscopic level with cryo-prepared and chemically fixed tissues. Polyclonal antibodies (Abs), developed in this study to the native protein (native Abs), removed or much decreased the background difficulties that persisted with the use of Abs to denatured SCA and SCA peptide Abs. The polyclonal Abs localize SCA to the wall and the cytoplasm of specialized pistil epidermal cells, the style and stigma transmitting tract epidermis. Pollen does not produce SCA protein yet in vivo grown pollen tubes in cryo-preparations showed cytoplasmic localization of SCA. In vitro grown pollen tubes, cultured in growth medium containing SCA, showed strong label at the pollen tube tip and back from the tip in the wall. Transmission electron microscopy data using in vivo grown pollen tubes confirmed localization in the cytoplasm and wall though localization of SCA inside the tube cell cytoplasm in these preparations was not definitive because of the high background level in the controls.