Vascular plants have evolved shoot apical meristems (SAMs), whose structures differ among plant groups. To clarify the evolutionary course of the different structural types of SAMs, we compared plasmodesmatal networks in the SAMs for 17 families and 24 species of angiosperms, gymnosperms, and pteridophytes, using transmission electron microscopy (TEM). The plasmodesmata (PD) in almost all cell walls in median longitudinal sections of SAMs were counted, and the PD density per unit area was calculated for each cell wall. Angiosperm and gymnosperm SAMs have low densities, with no difference between stratified (tunica-corpus) and unstratified structures. SAMs of ferns, including Psilotum and Equisetum, have average densities that are more than three times higher than those of seed plants. Interestingly, microphyllous lycopods have both the fern and seed-plant types of PD networks; Selaginellaceae SAMs with single apical cells have high PD densities, while SAMs of Lycopodiaceae and Isoetaceae with plural initial cells have low PD densities, equivalent to those of seed plants. In summary, PD networks are strongly correlated to SAM organizations-SAMs with single and plural initial cells have the fern and seed-plant types of PD, respectively. The two SAM organizations may have evolved separately in lycophytes and euphyllophytes and may be associated with gain or loss of the ability to form secondary PD.

In ovules of Pinus densiflora, pollen tubes elongate 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. Vacuoles were collapsed, chromatin was condensed, and mitochondria 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 results 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.

Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.

LIM14, originally identified as a lily gene associated with microsporogenesis, encodes a protein which has two distinct domains, one with glycine-serine repeats and the other with a hydrophobic signal peptide at the N-terminus. The putative LIM14 protein, however, is distinct from the glycine-rich cell wall proteins which have been described before. RNA analyses indicated that the LIMN transcript is specifically detected in the anther from zygotene to young pollen stage. By using antibodies raised against recombinant LIM14 protein, we detected antherspecific 15 kDa protein. Immunofluorescence microscopy demonstrated that the LIM14 protein is associated with starch grains in the anther wall cells just prior to microspore mitosis and then accumulates at a higher level with the starch grains of immature pollen. We tagged LIM14 with the GUS and GFP reporter genes and introduced them into tobacco BY-2 cells. Analysis of the transformed cells revealed that the chimeric proteins are functional and specifically targeted to plastids. These results indicate that LIM14 is an anther-specific protein that may play a role in starch accumulation and amyloplast differentiation during anther development and pollen formation.Key words: Anther development -Glycine-rich proteinLilium longiflorum -Reporter genes -Starch grain.Pollen development in angiosperms is a highly regulated process which is dependent on interaction between sporophytic and gametophytic tissues of the anther. The structural details of pollen development are quite uniform in most species studied. During male meiosis each pollen mother cell (microsporocyte) undergoes two nuclear divisions resulting in the production of tetrads of four haploid microspores. Multiple cytological and molecular events are involved in the progression of microspores through the pollen developmental pathway, resulting in the formation of mature pollen grains. Other anther cell-types,

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