Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants

Tanaka, Ichiro; Nakamura, Sumio; Miki-Hirosige, H.

doi:10.1002/mrd.1120240403

Cited by 33 publications

(13 citation statements)

References 39 publications

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“…Pollen protoplasts, which have recently been isolated from pollen grains of several angiosperms (Tanaka et al 1987;Zhou 1989a), have already proved useful in the isolation of generative cells (Tanaka 1988), the observation of pollen nuclei and cytoplasm (Tanaka et al 1989), culture experiments to induce cell division (Zhou 1989b), and fusion studies (Ueda et al 1990). In the present study, the organization of actin filaments and MTs was examined during culture and development of pollen protoplasts.…”

Section: Discussionmentioning

confidence: 99%

Organization of the actin and microtubule cytoskeleton preceding pollen germination

Tanaka

Wakabayashi

1992

Planta

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Cultured pollen protoplasts of Lilium longiflorum Thunb. were used to study, with the aid of rhodamine-phalloidin staining and immunofluorescence, changes in the organization of actin filaments and microtubules (MTs) during cell-wall regeneration, transition of cell shape from spherical to ellipsoid, growth in cell size, and pollen-tube development. In freshly isolated protoplasts, an extensive network of minute actin filaments and disorganized MTs was observed in the central cytoplasm of the vegetative cell. After regeneration of the cell wall, cortical actin filaments and MTs gradually became organized but their orientation remained random. At 5-6 d of culture, when the protoplasts changed from a spherical to an ellipsoid shape, the cortical actin filaments were aligned almost in parallel, extending between two opposite foci, and in the growing ellipsoidal cell thick actin bundles became oriented transversely to the long axis of the cell. Following the ordering of the cortical actin filaments the cortical MTs also became arranged parallel to the actin filaments. After 8-10 d of culture, when the protoplasts germinated and formed a pollen tube, both actin filaments and MTs entered the tube. Cytochalasin B and colchicine added to the medium from the beginning of the protoplast culture inhibited the organization of the actin and MT cytoskeleton, and addition of the drugs from day 5 of culture onwards destroyed the organization and arrangement of the cytoskeleton. Disruption of the actin filaments by cytochalasin B resulted also in a disruption of the MT organization and arrangement, whereas disruption of the MTs by colchicine had little effect on the organization and arrangement of the actin filaments. Both drugs blocked pollen germination in the cultured protoplasts. From these observations it is assumed that the arrangement of actin filaments plays a predominant role in pollen germination, but that the arrangement of the MTs is also essential for pollen germination through maintaining cell polarity established by the actin arrangement.

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Section: Discussionmentioning

confidence: 99%

Organization of the actin and microtubule cytoskeleton preceding pollen germination

Tanaka

Wakabayashi

1992

Planta

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“…Pollen protoplasts were prepared by the previously described procedure (Tanaka et al 1987). Immunofluorescence staining was carried out by the previously described method (Tanaka et al 1989) with minor modifications. Isolated protoplasts were fixed in methanol at -20~ for 15 min and were then treated with 0.5% (v/v) Triton X-100 in PBS for 15 min at room temperature.…”

Section: Preparation Of Antisera and Immunoblotting Gel Pieces Contamentioning

confidence: 99%

Male gametic nucleus-specific H2B and H3 histones, designated gH2B and gH3, in Lilium longiflorum

Uéda

Tanaka

1995

Planta

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Abstract. Two proteins that resemble core histones and might be specific to the male gametic (generative) nucleus within the pollen of Lilium longiflorum Thumb. (originally designated p22.5 and p18.5; K. Ueda and I. Tanaka, 1994, Planta, 192, 446452) were characterized biochemically and immunochemically. Patterns of digestion of p22.5 and p 18.5 by Staphylococcus aureus V8 protease closely resembled those of somatic histones H2B and H3, respectively. However, peptide fragments that were unique to p22.5 or p18.5 were also detected. Antibodies raised against these proteins did not cross-react with any somatic histones. These results indicate that p22.5 and p18.5 are different from somatic histones in terms of primary structure. Analysis of their amino-acid compositions revealed that p22.5 is a moderately lysine-rich protein while p18.5 is an arginine-rich protein. From these results, we conclude that p22.5 is a variant of histone H2B and p18.5 is a variant of histone H3. Immunofluorescence staining of pollen grains using the specific antibodies revealed that both p22.5 and p18.5 are only present in the generative cell nucleus and are not to be found in the vegetative cell nucleus. This study demonstrates that (i) specific histone variants are present in the male gametic nucleus of a higher plant, as they are in the sperm nucleus of animals, and (ii) distinct differences in histone composition exist between the nuclei of generative and vegetative cells in pollen. These novel histones (p22.5 and p18.5), specific to male gametic nuclei, have been designated gH2B and gH3, respectively.

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“…For instance, the isolated GCs also show MT rearrangement from axial bundles to the network pattern through an intermediate form. The difference is that the in vivo development is a natural process with gradual and smooth changes, whereas artificial isolation causing a sudden change of the environment may result in a certain degree of MT depolymerization (Tanaka et al 1989;Zhou et al 1990). …”

Section: Discussionmentioning

confidence: 97%

“…1988;Palevitz and Cresti 1989;Pierson 1989;Taylor et al 1989;Xu et al 1990;Zhou et al 1990;Zhu and Liu 1990). More recently, microtubular changes in GCs artificially isolated from mature pollen grains have been dealt with (Zhou et al 1988;Tanaka et al 1989;Zhou et al 1990). In some of these investigations, GCs inside mature pollen grains were also observed for MT arrangement.…”

Section: Introductionmentioning

confidence: 98%

Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

Zhou

Hong-yuan

1991

Sexual Plant Reprod

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Summary.The three-dimensional organization of microtubules in generative cells during their development in pollen grains of Hippeastrum vittatum and the dynamic changes that occur were studied by collecting large quantities of fixed and isolated generative cells for immunofluorescence microscopy. The framework configuration and the arrangement pattern of the microtubule organization was investigated. The microtubule framework changed in shape from being spherical at an early stage to being long spindle-shaped at maturity: various transitional forms were observed: ellipsoidal, pear-shaped and short spindle-shaped. The microtubule arrangement making up this framework changed correspondingly from the original network, which was random in distribution, to axially oriented long bundles via an intermediate pattern composed of a mixture of networks with long bundles. However, cells with the same framework configuration might be heterogeneous in microtubule arrangements.

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Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants

Cited by 33 publications

References 39 publications

Organization of the actin and microtubule cytoskeleton preceding pollen germination

Organization of the actin and microtubule cytoskeleton preceding pollen germination

Male gametic nucleus-specific H2B and H3 histones, designated gH2B and gH3, in Lilium longiflorum

Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

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