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Immunogold labelling revealed the presence of lipoxygenase (LOX) in different parts and types of anther cells of Gagea lutea. LOX was found in the cytoplasm and close to ER elements in epidermal and endothecial cells, and close to the cell walls of the latter. The positive immunoreaction to LOX was less intense in the middle layers and the loculus of the anther, where single immunogold particles were concentrated at the cell walls of these layers and in the protoplast masses, in vacuoles, close to mitochondria, inside plastids, and in the liquid of the anther cavity. LOX occurred in the cytoplasm and around ER elements of pollen grains as well as in the exine layer, particularly in contact regions between the outer and inner exine layers. The correlations between LOX localization in different anther cells and the functioning of particular anther parts are discussed.
Karyological processes of differentiation of the suspensor of Gagea lutea (L.) Ker Gawl. were compared with the development of the embryo proper. The zygote divides into the smaller apical cell and the bigger basal cell, which becomes the basal cell of the suspensor. The mature suspensor consists of a huge basal cell and a few chalazal cells. The nuclear DNA content of the suspensor basal cell attains a high degree of ploidy, up to 128C. Nuclei with the highest ploidy level of 128C were found only in fully differentiated basal cells of more than 20-celled embryos. During polyploidization, the volume of the nuclei increased, and changes in the chromatin structure of polyploid nuclei were noted. With increasing levels of ploidy, polytene chromosomes were observed in the suspensor nucleus. Changes in DNA content, nucleus size and chromatin structure point to endoreduplication as the mechanism of polyploidization of the suspensor in Gagea lutea.
The microtubular cytoskeleton in dividing microsporocytes and developing pollen grains of Gagea lutea (L.) Ker.-Gaw. (Liliaceae) was investigated with a modified indirect immunofluorescence method. Meiotic and mitotic stages were identified by DAPI staining. The microtubular cytoskeleton was compared in plants originating from natural localities and others grown in the laboratory. In natural conditions, microsporocytes and pollen grains of wild early-spring Gagea lutea plants are subjected to abiotic factors including cold exposure and lack of water. The persistent influence of these factors can disturb microtubular cytoskeleton functioning. The following disturbances were observed in the course of microsporogenesis and pollen development: abnormal chromosome configurations in the metaphase of meiosis I; abnormally divided dyads with irregular, radial microtubule systems around the nuclei; the formation of differently sized microspores with irregular shapes, and irregular division; and the formation of pollen grains with vacuoles abnormal for their development stage. Similar kinds of disturbances were observed after 1.5 months of cold treatment (4°C) and drying in the laboratory. These abiotic factors simulated in laboratory conditions caused more disturbances in the course of microsporogenesis and produced more frequent defective pollen grains than in the sample that had experienced cold and drying in natural conditions.
The distribution of plastids at the time of microspore and pollen grain development in Gagea lutea (L.) Ker.-Gaw. was analyzed using electron microscopy. It was shown that plastids are not transmitted to the forming generative cell of this species during microspore division. At the vacuolate microspore stage, preceding division, the microspore nucleus takes an acentric position and the plastids gather at the opposite side of the cell. In the highly polarized microspore at prophase of mitosis, all plastids are aggregated at one side of the nucleus, whereas mitochondria are dispersed throughout the cytoplasm. Numerous profiles of endoplasmic reticulum (ER) are present between the clustered plastids. Some of the ER profiles are attached by their ends to the outer membrane of plastid envelopes and join the distant plastids. The outer membrane of the microspore plastids may form long and thin evaginations contacting with other plastids. Microtubules are visible in plastid aggregations occasionally. In dividing microspores, long ER cisterns surround the area of the mitotic spindle and separate it from the region containing plastids. There are no plastids in the young generative cell: all plastids remain clustered in the region of the microspore that now forms the vegetative cell of the bicellular pollen grain. Later the connections between plastids and ER cisterns gradually disappear and plastids disperse in the cytoplasm of the whole vegetative cell. The results of our study are not sufficient to define the mechanism causing selective aggregation of plastids at the vegetative pole of the Gagea microspore, nor to say whether the microtubular cytoskeleton plays a role. However, the participation of ER in these processes, at least in holding the special arrangement of microspore plastids, seems certain.
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