Pollen Development And Programmed Cell Death During The Development Of Prothallial Cell In Picea Asperata And Pinus Tabulaeformis

Pollen development process together with dynamics of the starch grain and callose during pollen development in Picea asperata and Pinus tabulaejbrmis were observed by means of light microscope and fluorescent microscope. The degraded process of prothallial cells in P asperata and P tabulaeformis was further studied by transmission electron microscope, gel electrophoresis and terminal deoxynucleotidyl tranferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method. The main results were summarized as follows. The pattern of pollen development in P asperata and P tabuiaeformis was similar to that of other species in Pinaceae. It took only about 20 days from the stage of microspore mother cell (MMC) to the pollen shedding time. Before undergoing meiosis, the MMCs separated from each other but still remained connection in some parts. During the meiosis process, the starch grains and the calloses changed regularly. From metaphase-I to telophase-I, the starch grains aggregated gradually in the equatorial region. At the tetrad stage, the starch grains mainly distributed in the place where the cell wall and the cytoplasm separated. The peripheral calloses in the equational region began to increase gradually toward the center of the MMC until the cytoplasm transformed into four microspores. Cytokinesis following meiosis was simultaneous, and tetrad was tetrahedral. The microspore with two small sacci contained many starch grains when they were released from the tetrad. As the microspore developed, its volume increased, vacuoles formed and the nucleus moved toward one side. The microspore underwent an asymmetric division, then formed a big central cell and a small prothallial cell, while the former soon further gave rise to the antheridial initial and the second prothallial cell. After the formation of prothallial cell, callose was 58 observed on the cell wall between the prothallial cell and the central cell or the antheridial initial. Subsequently, the callose disappeared as the prothallial cells degenerated. The antheridial initial gave rise to a tube cell and a generative cell. In this paper, three methods were used to study the degradation of the prothallial cells of P asperata and two methods were used to P tabulaeformis. The condensed chromatin in the nuclei of the degraded prothallial cells was shown by telescope electron microscopy; the nucleus and the vacuole might be the last organelles disappeared during the degradation process. These features are similar to that observed in animals and other plants. DNA ladders, resulting from the cleavage of nuclear DNA into oligonucleosomal fragments in degrading prothallial cells, were detected by gel electrophoresis. And the intra-nucleosomal DNA cleavage was further demonstrated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method. These results revealed that the degradation of the prothallial cells was a typical programmed cell death (PCD). Moreover, the pollen shed from the mature anthers are in large quantities and they can be easily collected for both biochemical and molecular analyses. Therefore, the development of prothallial cells in coniferous pollen might be served as a natural model system for further study on molecular mechanism of PCD.