Possible Involvement of Calmodulin and the Cytoskeleton in Electrofusion of Plant Protoplasts

Abe, Shunnosuke; Takeda, Jun

doi:10.1104/pp.81.4.1151

Cited by 14 publications

(7 citation statements)

References 28 publications

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“…Therefore, a recovery of fusion frequency after washing the La3 + -treated protoplasts is attributable to a recovery of dielectrophoresis. In the presence of 1 mm La3 +, dielectrophoresis of protoplasts was inhibited almost completely, while tight clusters of several protoplasts increased as reported earlier (2). Fusion efficiency was very low even after prolonged incubation which caused a considerable increase of aggregation (>50%).…”

Section: Reversibility Of the Effects Of La3+ On Dielectrophoresis Andsupporting

confidence: 48%

“…This indicates that the inhibitory effect on the initial induction and the promotive effect on the development of fusion are exerted by different mechanisms. These promotive effects might arise from a modification of the cytoskeleton, since Ln3 + (23) and other multivalent cations (21) are reported to interact with it, and effects similar to those of cytochalasin B have been reported (2). The possibility that these trivalent cations are taken up during electroporation and exert their promotive effects internally cannot be excluded.…”

Section: Discussionmentioning

confidence: 96%

“…In our earliest study of electrofusion (19) and in related studies (1)(2)(3), the importance of calcium ions and a change of the membrane state into a fusible state in induction of electrofusion have been pointed out. We used Ca2+ in order to stabilize and enhance electrofusion.…”

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confidence: 99%

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Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Abe¹,

Takeda²

1988

Plant Physiol.

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When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 mil-limolar lanthanum ion (La3+) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N,N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl2), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La3+-treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La3+-treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La3+ after washing without EDTA and released most of the bound La3+ by washing with 1 millimolar EDTA. This tightly bound La3+ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La3+-treated protoplasts after washing. {-potentials of protoplasts were-39.0 ± 3.2 millivolts,-16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La3+ (I= 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La3+. These effects of La3+ on {-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La3+ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La3+ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently. In our earliest study of electrofusion (19) and in related studies (1-3), the importance of calcium ions and a change of the membrane state into a fusible state in induction of electrofusion have been pointed out. We used Ca2+ in order to stabilize and enhance electrofusion. The effectiveness of Ca2 + on electrofusion was recently confirmed with plant (7, 14, 17, 20, 21) and animal cells (15). On the other hand, a low fusion efficiency, compared with a weakly conductive pure osmoticum medium, has been reported because addition of Ca salts increases conductivity of media (25). In mesophyll protoplasts from peas, Ca2+ reduces the threshold for electrofusion (7). In protoplasts from tobacco leaf cells and cultured potato cells, fusion frequencies are increased by the addition of Ca2+ (20). An absolute requirement of Ca2 + in elec-trofusion is reported with animal cells (15). In some cases, Mg2+ I Supported by a grant from the Ministry of Education, Science and Culture of Japan. ions also facilitate electrofusion, in addition to or instead of Ca2 + (25). These stimulative effects of Ca2+ on electrofusion do not seem to be adequately explained by the reduction of the fluidity of the lipid...

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Section: Reversibility Of the Effects Of La3+ On Dielectrophoresis Andsupporting

confidence: 48%

Section: Discussionmentioning

confidence: 96%

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Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Abe¹,

Takeda²

1988

Plant Physiol.

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“…Both cytochalasin B and cytochalasin D have been reported to promote the process of spherulation of fusion products through the depolymerization of microfilaments in the cytoskeleton into actin monomers, following inhibition of elongation (Abe & Takeda 1986;Abe & Takeda 1989). From Table 2, it is evident that the two compounds did not have a particular effect on the efficiency of electrofusion of leaf mesophyll N. tabacum cv.…”

Section: ( D) Overall Viabilities (%) O F Fusion Products 24 H After mentioning

confidence: 75%

“…Although the effects of a wide range of chemical fusion facilitators on electrofusion have been reported (Abe & Takeda 1986;Ruzin & McCarthy 1986;Christov & Vaklinova 1987;Abe & Takeda 1989), no information on the comparative effects of the chemicals has been published. It was for this reason that we characterized the effects of polyamines, cytochalasins, proteases, lipase, prostaglandins, lecithin and two Ca 2+ ion sources for their effects on five electrofusion and overall survival attributes of fusion-derived products.…”

Section: Introductionmentioning

confidence: 99%

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Matibiri

Mantell

1995

Plant Cell Tiss Organ Cult

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Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L. shoots were subjected to electrofusion.Dielectrophoresis was induced by an AC field of 50 V cm -1 inter-electrode distance and 0.5 MHz oscillation frequency. Fusion was effected by two 0.7 kV cm-1 DC pulses, each of 50 Us duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectrophoresis efficiencies (percentages ofprotoplasts that made contact with at least one other protoplast under the AC field), fusion efficiencies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion processes), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-derived cells that had undergone division). The various attributes assessed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on different days. Relative to the control treatment, only the Ca 2+ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis efficiencies. Polyamines, cytochalasins and Ca 2+ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg 1-1, the cytochalasins B and D, and Ca 2+ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca 2+ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin and CaC12 treatments, overall plating efficiencies were reduced by treatment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treatment were obtained using prostaglandin F2a, lecithin and CaC12.2H20 treatments. The implications of the results are discussed.

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Uptake of Fungal Protoplasts by Plant Protoplasts

Lynch

Isaac

Collin

1989

Plant Protoplasts and Genetic Engineering II

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Possible Involvement of Calmodulin and the Cytoskeleton in Electrofusion of Plant Protoplasts

Cited by 14 publications

References 28 publications

Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Uptake of Fungal Protoplasts by Plant Protoplasts

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Possible Involvement of Calmodulin and the Cytoskeleton in Electrofusion of Plant Protoplasts

Cited by 14 publications

References 28 publications

Effects of La3+ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Effects of La3+ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Uptake of Fungal Protoplasts by Plant Protoplasts

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Product

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Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts