Programmed cell death of Pinus nucellus in response to pollen tube penetration

In ovules of Pinus densiflora, pollen tubes elon-eate and branch into the nucellar tissue in the direction of the female gametophyte. After pollination, nucellar cells located around the pollen grain and tube die off. We showed here that the nuclei of the nucellar cells were stained by TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling). The number of TUNEL-positive cells increased during pollen tube growth. The tips of pollen tube branches protruded into the nucellar cells to form a convex-concave junction. At this junction, the cell membrane of nucellar cells was separated from the cell wall and the protoplast shrank. Small vesicles and amorphous materials were released from the protoplast into the space between the cell membrane and wall. Vacu-oles were collapsed, chromatin was condensed, and mito-chondria and plastids were deteriorated in the shrunken protoplast. Agarose gel analysis of DNA isolated from the ovules showed a DNA ladder, suggesting that the nuclear DNA had undergone internucleosomal cleavage. These re-sults suggest that nucellar cells undergo programmed cell death in response to pollen tube penetration with some features resembling apoptosis and other features peculiar to nucellar cells.