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Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana.

Kawashima T, Maruyama D, Shagirov M, Li J, Hamamura Y, Yelagandula R, Toyama Y, Berger F - Elife (2014)

Bottom Line: Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus.We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin-myosin interactions are also involved in male gamete nucleus migration.The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore.

ABSTRACT
In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin-myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.

No MeSH data available.


Related in: MedlinePlus

F-actin is required for egg cell fertilization.(A) Cartoon of Arabidopsis mature ovule. cc, central cell; cz, chalaza; ec, egg cell; mp, micropyle; sy, synergid. (B–D) Egg cell actin cables (B) become disassembled in LatA treatment (C) and in DN-ACTIN (D). (E and F) Successful fertilization marked by decondensation of the sperm cell chromatin (ssc, red) into the egg cell nucleus (dashed oval) (E), resulting in a normal embryo in WT (F). (G and H) Egg cell expressing DN-ACTIN shows arrests in sperm cell nuclear migration (G) and embryo development (H). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.003
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fig1: F-actin is required for egg cell fertilization.(A) Cartoon of Arabidopsis mature ovule. cc, central cell; cz, chalaza; ec, egg cell; mp, micropyle; sy, synergid. (B–D) Egg cell actin cables (B) become disassembled in LatA treatment (C) and in DN-ACTIN (D). (E and F) Successful fertilization marked by decondensation of the sperm cell chromatin (ssc, red) into the egg cell nucleus (dashed oval) (E), resulting in a normal embryo in WT (F). (G and H) Egg cell expressing DN-ACTIN shows arrests in sperm cell nuclear migration (G) and embryo development (H). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.003

Mentions: To investigate the requirement and function of F-actin during fertilization, we expressed the fluorescent reporter Lifeact-Venus (Riedl et al., 2008; Era et al., 2009) under the control of the egg cell-specific EC1 promoter (Sprunck et al., 2012) to visualize the actin cytoskeleton in the egg cell (Figure 1A,B). Lifeact-Venus marked cables were disassembled after treatment with the actin polymerizing inhibitor Latrunculin A (LatA; Figure 1C). Pharmacological analysis by applying inhibitor drugs is useful to dissect out the cytoskeleton function at the cellular level. However, treatment with actin polymerization inhibitors disrupts functions in all cells when applied to tissues such as ovules and thus prevents the analysis on specific cytoskeleton functions in a specific cell-type. To overcome this problem, the semi-dominant negative ACTIN transgene (DN-ACTIN;Kato et al., 2010) was introduced to disrupt F-actin specifically in female gametes. DN-ACTIN contains one amino acid substitution in the hydrophobic loop of Arabidopsis ACT8, which causes instability and fragmentation of actin filaments, leading to incomplete yet strong disruption of actin cytoskeleton (Kato et al., 2010). Consistent with the effect of DN-ACTIN reported previously, the filamentous structures shown in the wild-type (WT) egg cell became much shorter and generated aggregates in the egg cell expressing DN-ACTIN (Figure 1D). In WT plants, fertilization leads to karyogamy followed by decondensation of the chromatin from the male nucleus (Figure 1E; Ingouff et al., 2007). Egg cell fertilization initiates embryo development while the fusion of the other sperm cell with the central cell leads to endosperm development (Figure 1A,F). By contrast, fertilization of the egg cell expressing DN-ACTIN failed as the sperm nucleus did not fuse with the egg cell nucleus and the sperm chromatin remained condensed (Figure 1G; Line 1, 35% defects in DN-ACTIN [n = 104] compared to 0% defects in WT [n = 98]). Karyogamy was prevented only in the egg cell expressing DN-ACTIN but not in the central cell, resulting in a seed containing endosperm without an embryo [Figure 1H; Line 1, 27% defects in DN-ACTIN (n = 110) compared to 0% defects in WT (n = 389)]. Taken together, these results suggest that actin cytoskeletons are required for egg cell fertilization. Consistently, other independent transgenic lines showed similar seed developmental arrest [Line 2, 20% defects (n = 125); Line 3, 22% defects (n = 114)]. Not all ovules of DN-ACTIN expressing lines showed the fertilization defect, likely because a certain fraction of actin filaments was still functional.10.7554/eLife.04501.003Figure 1.F-actin is required for egg cell fertilization.


Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana.

Kawashima T, Maruyama D, Shagirov M, Li J, Hamamura Y, Yelagandula R, Toyama Y, Berger F - Elife (2014)

F-actin is required for egg cell fertilization.(A) Cartoon of Arabidopsis mature ovule. cc, central cell; cz, chalaza; ec, egg cell; mp, micropyle; sy, synergid. (B–D) Egg cell actin cables (B) become disassembled in LatA treatment (C) and in DN-ACTIN (D). (E and F) Successful fertilization marked by decondensation of the sperm cell chromatin (ssc, red) into the egg cell nucleus (dashed oval) (E), resulting in a normal embryo in WT (F). (G and H) Egg cell expressing DN-ACTIN shows arrests in sperm cell nuclear migration (G) and embryo development (H). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.003
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4221737&req=5

fig1: F-actin is required for egg cell fertilization.(A) Cartoon of Arabidopsis mature ovule. cc, central cell; cz, chalaza; ec, egg cell; mp, micropyle; sy, synergid. (B–D) Egg cell actin cables (B) become disassembled in LatA treatment (C) and in DN-ACTIN (D). (E and F) Successful fertilization marked by decondensation of the sperm cell chromatin (ssc, red) into the egg cell nucleus (dashed oval) (E), resulting in a normal embryo in WT (F). (G and H) Egg cell expressing DN-ACTIN shows arrests in sperm cell nuclear migration (G) and embryo development (H). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.003
Mentions: To investigate the requirement and function of F-actin during fertilization, we expressed the fluorescent reporter Lifeact-Venus (Riedl et al., 2008; Era et al., 2009) under the control of the egg cell-specific EC1 promoter (Sprunck et al., 2012) to visualize the actin cytoskeleton in the egg cell (Figure 1A,B). Lifeact-Venus marked cables were disassembled after treatment with the actin polymerizing inhibitor Latrunculin A (LatA; Figure 1C). Pharmacological analysis by applying inhibitor drugs is useful to dissect out the cytoskeleton function at the cellular level. However, treatment with actin polymerization inhibitors disrupts functions in all cells when applied to tissues such as ovules and thus prevents the analysis on specific cytoskeleton functions in a specific cell-type. To overcome this problem, the semi-dominant negative ACTIN transgene (DN-ACTIN;Kato et al., 2010) was introduced to disrupt F-actin specifically in female gametes. DN-ACTIN contains one amino acid substitution in the hydrophobic loop of Arabidopsis ACT8, which causes instability and fragmentation of actin filaments, leading to incomplete yet strong disruption of actin cytoskeleton (Kato et al., 2010). Consistent with the effect of DN-ACTIN reported previously, the filamentous structures shown in the wild-type (WT) egg cell became much shorter and generated aggregates in the egg cell expressing DN-ACTIN (Figure 1D). In WT plants, fertilization leads to karyogamy followed by decondensation of the chromatin from the male nucleus (Figure 1E; Ingouff et al., 2007). Egg cell fertilization initiates embryo development while the fusion of the other sperm cell with the central cell leads to endosperm development (Figure 1A,F). By contrast, fertilization of the egg cell expressing DN-ACTIN failed as the sperm nucleus did not fuse with the egg cell nucleus and the sperm chromatin remained condensed (Figure 1G; Line 1, 35% defects in DN-ACTIN [n = 104] compared to 0% defects in WT [n = 98]). Karyogamy was prevented only in the egg cell expressing DN-ACTIN but not in the central cell, resulting in a seed containing endosperm without an embryo [Figure 1H; Line 1, 27% defects in DN-ACTIN (n = 110) compared to 0% defects in WT (n = 389)]. Taken together, these results suggest that actin cytoskeletons are required for egg cell fertilization. Consistently, other independent transgenic lines showed similar seed developmental arrest [Line 2, 20% defects (n = 125); Line 3, 22% defects (n = 114)]. Not all ovules of DN-ACTIN expressing lines showed the fertilization defect, likely because a certain fraction of actin filaments was still functional.10.7554/eLife.04501.003Figure 1.F-actin is required for egg cell fertilization.

Bottom Line: Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus.We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin-myosin interactions are also involved in male gamete nucleus migration.The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore.

ABSTRACT
In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin-myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.

No MeSH data available.


Related in: MedlinePlus