Computer-assisted reconstruction of the male germ unit in pollen ofBrassica campestris

McConchie, C. A.; Jobson, S.; Knox, R. B.

doi:10.1007/bf01273701

Cited by 62 publications

(19 citation statements)

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“…Similar physical associations between the vegetative nucleus and the sperm cell pair have recently been reported in several species (13,20) and are termed 'male germ unit' (MGU) by Dumas et al (5) to emphasize the male DNA transmission as a whole in the double fertilization. In addition, Russell (15) pointed out the preferential fusion of the rich plastid sperm cell (SC2) with the egg in Plumbago.…”

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confidence: 55%

Male Germ Unit Isolation from Three Tricellular Pollen Species: Brassica oleracea, Zea mays, and Triticum aestivum

Matthys‐Rochon¹,

Vergne²,

Detchepare³

et al. 1987

Plant Physiol.

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ABSTRACIA technical procedure is described to follow the in vitro release of the 'male germ unit' and the sperm cells from three tricellular pollen species (Brassica, Zea, and Triticum). The condition of the sperm cell was controlled using light microscopy. In addition, for the first time, the sperm cells viability has been checked by the fluorochromatic reaction test. These preliminary results indicate that this procedure appears to be a prerequisite for the successful preparation of purified viable sperm cells.In flowering plants, the sexual process of double fertilization is still relatively unknown especially the mechanisms of cell fusions. Two sperm cells (SCl and SC2)2 produced by the pollen grain are required: one fuses with the egg to form the embryo and the second fuses with the central cell to give rise to the nutritive endosperm. Today it is a challenge to understand the role played by each sperm cell. With that in mind, attempts have been made to prepare naked pollen gametophytes (sporoplasts) (12) and sperm cell nuclei (11).On the other hand, Russell (16,17) (3) were used. The details of the technical conditions are reported in Table I. RESULTS AND DISCUSSIONThe pollen species used in this work are tricellular: a vegetative cell containing two sperm cells whose nuclei may be visualized in situ by the DNA fluorochrome DAPI (Fig. 1). The isolation ofsperm cells first involves the finding ofan appropriate medium and technique to break the thick pollen wall. To obtain them free of damage, the release of intact sperm cells must be controlled throughout the isolation procedure. Finally their viability has to be verified and checked.Thus pollen grains were incubated in a BK sucrose medium to induce a light osmotic shock. For Zea and Brassica pollen a gentle grinding is coupled with the osmotic shock. In Zea pollen congestion of organelles prevents accurate observation of sperm cells which led us to find the following convenient and rapid method: using a light microscope equipped with both fluorescence and phase contrast systems to simultaneously detect the nuclei with a DNA probe (EB or DAPI) and to determine the cellular state by phase contrast microscopy. Under these conditions the release of sperm cells follows a remarkable 'time table. ' When pollen grains discharge their contents, the vegetative nucleus and the two sperm cells appear simultaneously. These three subunits are linked to form a structured set, which has previously been termed the MGU (5), demonstrated from electron microscopic data. We present the first light microscopic observation ofthe in vitro MGU in Brassica (Fig. 2). In Triticum and Zea pollens the abundance ofstorage products (starch grains) makes the MGU structure more difficult to liberate in good condition. After pollen discharge, the two sperm cells separate from the vegetative nucleus. Then they appear as elongated cells with a reduced 'active' cytoplasm, connected with a membranous tract (Fig. 3, a and b

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confidence: 55%

Male Germ Unit Isolation from Three Tricellular Pollen Species: Brassica oleracea, Zea mays, and Triticum aestivum

Matthys‐Rochon¹,

Vergne²,

Detchepare³

et al. 1987

Plant Physiol.

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“…Mature pollen, rehydrated in Brewbaker and Kwack media with 20% sucrose (Brewbaker and Kwack 1963), and developing anthers were prepared for electron microscopy by modifying the procedure described in McConchie et al (1985). Material was fixed at room temperature for 4 h, in 2.5% glutaraldehyde in 0.03 M 1,4-piperazinediethanesulphonic acid (Pipes) buffer (pH 7.4), 12% sucrose and washed with Pipes buffer (20% sucrose), dehydrated by ethanol series, and then post-fixed with 1% OsO4 in Pipes buffer (20 % sucrose).…”

Section: Methodsmentioning

confidence: 99%

The interrelationship between the accumulation of lipids, protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Evans

Taylor

Singh

et al. 1992

Planta

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Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.

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“…In the MGU, one sperm cell is connected to the vegetative nucleus via a "tail," or cell extension, containing forked arrays of microtubules. This tail penetrates the highly convoluted vegetative nucleus (McConchie et al, 1985), whereas the two sperm cells are enclosed within a vegetative cell membrane-bound compartment, and are linked by a transverse cell wall and evaginations of their plasma membranes (Dumas et al, 1984a(Dumas et al, , 1984bCharzinska et al, 1989). In mature bicellular pollen systems, and in tricellular pollen of monocotyledonous species such as maize (Zea mays), the MGU is not observed in mature pollen, but assembly occurs early during pollen tube growth (Hu and Yu, 1988;Rougier et al, 1991).…”

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Genetic Control of Male Germ Unit Organization in Arabidopsis

Lalanne

Twell

2002

Plant Physiology

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In flowering plants, the vegetative nucleus and the two sperm cells are proposed to form a functional assemblage, the male germ unit (MGU). Here, we describe the developmental pathway of MGU assembly in Arabidopsis and report two classes of mutations that affect the integrity and/or the positioning of the MGU in the mature pollen grain. In germ unit malformed (gum) mutants, the vegetative nucleus is positioned adjacent to the pollen grain wall, separate from the two sperm cells, whereas in MGU displaced (mud) mutants, the intact MGU is displaced to the pollen grain wall. mud and gum mutants correspond to male-specific gametophytic mutations that also reduce pollen fitness. Genetic mapping showed that the gum1 and gum2 mutations are genetically linked, possibly allelic, whereas the mud1 and mud2 mutations correspond to two unlinked loci mapping on different chromosomes. The hierarchical relationship between mud and gum mutations was investigated by phenotypic analysis of double mutants. gum1 appeared to act earlier than mud1 and mud2, affecting initial MGU assembly and its stability during pollen maturation. In contrast, mud1 and mud2 mutations appear to act only on MGU positioning during final maturation. From in planta analyses of pollen germination in mud and gum mutants, we conclude that the initial proximity and positioning of MGU components is not required for their entrance into the pollen tube, but the efficiency of MGU translocation is reduced.

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Computer-assisted reconstruction of the male germ unit in pollen ofBrassica campestris

Cited by 62 publications

References 10 publications

Male Germ Unit Isolation from Three Tricellular Pollen Species: Brassica oleracea, Zea mays, and Triticum aestivum

Male Germ Unit Isolation from Three Tricellular Pollen Species: Brassica oleracea, Zea mays, and Triticum aestivum

The interrelationship between the accumulation of lipids, protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Genetic Control of Male Germ Unit Organization in Arabidopsis

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