FERTILIZATION IN NICOTIANA-TABACUM - CYTOSKELETAL MODIFICATIONS IN THE EMBRYO SAC DURING SYNERGID DEGENERATION - A HYPOTHESIS FOR SHORT-DISTANCE TRANSPORT OF SPERM CELLS PRIOR TO GAMETE FUSION

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-s ubstituted and chemically fixed material in combination with immunoflu orescence and immunogold electron microscopy. Before fertilization, th e synergid is a highly polarized cell with dense longitudinally aligne d arrays of microtubules adjacent to the filiform apparatus at the mic ropylar end of the cell associated with major organelles. The cytoskel eton of the central cell is less polarized, with dense cortical microt ubules in the micropylar and chalazal regions and looser, longitudinal ly oriented cortical microtubules in the lateral region. In the synerg id and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microt ubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin fil aments are associated with microtubules and the cortex of the egg cell . At 48 h after pollination and before the pollen tube arrives, the on set of degeneration is evident in one of the two synergids: the electr on density of cytoplasmic organelles and the ground cytoplasm increase s and the nucleus becomes distorted. Although synergids otherwise rema in intact, the vacuole collapses and organelles degenerate rapidly aft er pollen-tube entry. Abundant electron-dense material extends from th e degenerated synergid into intercellular spaces at the chalazal end o f the synergid and between the synergids, egg and central cell. Rhodam ine-phalloidin and anti-actin immunogold labeling reveal that electron -dense aggregates in this region contain abundant actin forming two di stinct bands termed ''coronas''. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate t he access of male gametes to the egg and central cell for fusion.