Ultrastructural and immunocytochemical demonstration of gametophytic proteins in the pollen tube wall of the primitive gymnosperm Cycas

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Abstract
Ripe pollen of the primitive gymnosperm Cycas armstrongii germinates 8–15 days after sowing on a suitable artificial medium. During the pre-germination interval, the tube cell of the tricellular male gametophyte is actively engaged in the synthesis and export of polysaccharide material. This polysaccharide is laid down as a continuous microfibrillar stratum between the tube cell plasmalemma and the pollen wall intine. While the polysaccharide layer is forming, numerous evaginations arise as cytoplasmic extensions from the tube cell and penetrate the component. The structural link between the cell and the evaginations is eventually lost and the isolated fragments of cytoplasm immediately break up to form discrete particles. These particles remain within the channels previously occupied by the evaginations. At germination, the exine and intine layers of the pollen wall are disrupted and the tube cell emerges through the germinal aperture to establish the pollen tube. Polysaccharide deposition is resumed at about this stage, and consequently, a second microfibrillar layer begins to form beneath the first. The wall of the early pollen tube, therefore, has two distinct zones: the outer stratum is a loose superficial reticulum of fibrils and contains cytoplasmic inclusions; the inner stratum is structurally compact and is without inclusions. Throughout the period of tube development, the presence of protein, including the enzyme acid phosphatase, can be demonstrated in the outer wall zone with cytochemical methods. Immunofluorescent staining using pollen antiserum immunoglobulin G in an indirect technique with fluorescein isothyocyanate-labelled secondary antibody shows that one component in the pollen tube wall is immunologically related to the male gametophyte, and results from immunoelectron microscopy support the conclusion that this consists of the inclusions rather than the polysaccharide fabric of the tube wall itself. There are remarkable similarities between the cytoplasmic inclusions in the tube wall in Cycas and those found in the intine layer of flowering plant pollen. Both are of haplophase derivation, although the inclusions in the flowering plants are introduced via evaginations into the intine, the incipient pollen tube wall, at a much earlier stage in pollen development than they are in Cycas. The difference in the time of incorporation represents, perhaps, one more episode in the progressive evolutionary trend towards rapid pollen tube initiation and growth, and hence rapid fertilization, in spermatophyte pollen. This possibility is discussed, taking into account the view that the cycads and flowering plants are groups with a common ancestor.