Migration of sperm cells during pollen tube elongation in Arabidopsis thaliana: behavior during transport, maturation and upon dissociation of male germ unit associations

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Abstract
The promoter sequence of sperm-expressed gene, PzIPT isolated from the Svn (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.