Fertilization in Plumbago zeylanica: Gametic Fusion and Fate of the Male Cytoplasm

Abstract

Developmental phases surrounding the processes of gametic delivery and fusion were examined ultrastructurally in the reduced megagametophyte of Plumbago zeylanica, which lacks synergids. Gametic delivery occurs at the end of pollen tube growth and results in deposition of two male gametes, a vegetative nucleus, and a limited amount of pollen cytoplasm between the egg and central cell. Discharge of these materials from the tube is accompanied by loss of inner and outer pollen tube plasma membranes, loss of sperm-associated cell wall components, and disruption of the formerly continuous cell wall between the egg and central cell. The dispersion of egg cell wall components directly exposes female reproductive cell membranes to the unfused male gametes and pollen tube without disrupting gametic cell plasma membranes. Presence of unfused sperms within the female gametophyte appears to be a transitory phenomenon, lasting less than 5 min at the end of over 8½ hr of pollen tube growth. At the time of gametic deposition, plasma membranes of unfused sperm cells become directly appressed to plasma membranes of both the egg and central cell. Gametic fusion is initiated by a single fusion event between membranes of participating male and female cells, which is rapidly followed by subsequent, secondary fusion events between the same two cells at different locations along their surface. Gametic fusion results in the transmission of male gamete nuclei with co-transmission of nearly the entire sperm cytoplasmic volume and organellar complement, and it is possible to identify heritable male cytoplasmic organelles within both the incipient zygote and endosperm. Paternally originating plastids may be distinguished from maternal plastids by differences in morphology and staining characteristics, whereas paternal mitochondria may be distinguished from maternal mitochondria by populational differences in mitochondrial size which are statistically significant. Such observations further indicate that transmitted paternal mitochondria seem to remain viable, as judged by their ultrastructural appearance, and are transmitted exclusively by sperm cytoplasm rather than discharged pollen cytoplasm. The presence of anucleate, membrane-bounded cytoplasmic bodies between the egg and central cell are identifiable on the basis of their enclosed organelles and indicate that fragmentation of a small amount of the sperm cytoplasm associated with the vegetative nucleus commonly occurs. The presence and identification of sperm cytoplasmic organelles and associated membranes within female reproductive cells following gametic transmission represents strong evidence in support of the cellular basis of nuclear and cytoplasmic transmission during sexual reproduction in Plumbago.