Cell-wall development in freeze-fixed pollen: Intine formation of Ledebouria socialis (Hyacinthaceae)

The structure and development of the inner pectocellulosic pollen wall, the intine, was re-examined using high-pressure freezing with subsequent freeze substitution in Ledebouria socialis Roth, a monocotyledonous angiosperm. The bilayered intine is formed immediately after differentiation of the endexine. Similar to somatic cell walls, intine matrix substances originate from the Golgi apparatus and leave the cytoplasm via exocytosis. Exintine development starts with the apposition of intine matrix substances to the inner polysaccharide layer of the endexine (termed inner endexine), leading to irregular cell-wall ingrowths. Subsequently the inner endexine becomes intensely infiltrated with intine matrix substances; this process is interpreted as transformation of the inner endexine into intine. Along the aperture region, cell-wall matrix substances are unevenly deposited to such an extent that more or less radially oriented tubules filled with cytoplasm remain within the growing exintine. These tubules subsequently become cut off from the microspore cytoplasm by selective membrane fusions, leading to the incorporation of ground cytoplasm and ribosomes into the exintine. Exintine formation is completed prior to the first mitotic division of the pollen grain whereas the endintine is formed as a homogeneous thin layer after mitosis. Both transformation of the inner endexine by infiltration and passive incorporation of cytoplasm and ribosomes into the exintine by membrane fusions are novel features and are only observed in optimally freeze-fixed, freeze-substituted samples; general aspects of ultrastructure preservation in high-pressure-frozen, freeze-substituted plant cells are discussed as well. Modifications of the Golgi apparatus and post-Golgi-apparatus structures during pollen wall development are correlated with increasing and decreasing polysaccharide exocytosis, respectively. These evenls strictly coincide with the formation of morphologically and chemically different pollen wall layers and therefore seem to reflect the different deposition patterns of the predominant cell-wall polysaccharides.Abbreviations ER endoplasmic reticulum - FS freeze substitution - HPF high-pressure freezing - MS microspore(s) - PATAg periodic acid-thiocarbohydrazine-silver proteinate - PGS post-Golgi-apparatus structures - UA-Pb uranyl acetateleadI am grateful to Dr. Martin Müller (Institut für Zellbiologie, ETH-Zürich) for the kind permission to use the high-pressure freezer and the freeze-substitution unit at his laboratory. I wish to thank Prof. M. Hesse, Mag. M.G. Schlag (Institut für Botanik, Universität Wien) and Dr. I. Lichtscheidl (Institut für Pflanzenphysiologie, Universität Wien) for helpfull discussions. Thanks are also due to A. Glaser and W. Urbancik for excellent technical assistence and to the Stadtgärtnerei Zürich for providing the plant material. This work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.