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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.


The expression of neither constitutively-active ROP8 (CA-ROP8) nor dominant-negative ROP8 (DN-ROP8) under the control of the ROP8 promoter affect the F-actin structure (green) in the central cell.Autofluorescence marks the central cell outline (red line). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.013
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fig5s2: The expression of neither constitutively-active ROP8 (CA-ROP8) nor dominant-negative ROP8 (DN-ROP8) under the control of the ROP8 promoter affect the F-actin structure (green) in the central cell.Autofluorescence marks the central cell outline (red line). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.013

Mentions: We observed that, upon entry into the central cell, the sperm cell nucleus becomes surrounded by an increasing number of F-actin cables (Figure 3A–D), followed by the formation of a dynamic aster-like structure around the sperm cell nucleus (Figure 3E–H). This aster-like structure of F-actin was only observed around the sperm cell nucleus in the central cell after plasmogamy (100%, n = 48) and was never observed in unfertilized central cells (0%, n = 72). Consistently, the time-lapse imaging showed the presence of aster-like F-actin structure only during sperm cell nucleus migration [100% (n = 5); 0% in unfertilized ovule (n = 14)].The F-actin aster-like structure migrated together with the sperm cell nucleus towards the central cell nucleus (Video 1), suggesting the involvement of specific actin regulators in this process. In plants, F-actin assembly is regulated by formins and ARP2/3 complex under the control of plant-specific Rho-GTPases (ROP; Craddock et al., 2012; Henty-Ridilla et al., 2013). Transcriptional profiling (Borges et al., 2008; Le et al., 2010; Wuest et al., 2010; Belmonte et al., 2013) suggested that ROP8 is specifically active in the central cell and the endosperm (Figure 5A). Indeed, we found that the ROP8 promoter is specifically active in the central cell (Figure 5B) and the fusion protein Venus-ROP8 associates to the plasma membrane of the central cell (Figure 5C). Consistently, the ectopic expression of GFP-ROP8 also showed its association to the plasma membrane in root epidermal cells (Figure 5—figure supplement 1), indicating that ROP8 sub-localization is controlled by general machinery common between the gametophytic central cell and somatic root epidermal cell. To understand the function of ROP8 in the central cell during fertilization, we investigated F-actin organization in central cells expressing constitutively active and dominant-negative forms (Yang, 2002) of ROP8 (CA-ROP8 and DN-ROP8, respectively) under the control of the ROP8 promoter. Although F-actin structures in both CA-ROP8 and DN-ROP8 appeared similar to WT (Figure 5—figure supplement 2), only DN-ROP8 lines showed that the sperm cell chromatin in the central cell remained condensed and unfused with the central cell nucleus, when the sperm cell chromatin had already fused with the egg cell nucleus and appeared decondensed (Figure 5D; Line 1, 42% defects [n = 98], Line 2, 36% defects [n = 102]). This phenotype was similar to the fertilization defect observed in central cells expressing DN-ACTIN (Figure 4).10.7554/eLife.04501.011Figure 5.ROP8 is specific to the central cell and involved in 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)

The expression of neither constitutively-active ROP8 (CA-ROP8) nor dominant-negative ROP8 (DN-ROP8) under the control of the ROP8 promoter affect the F-actin structure (green) in the central cell.Autofluorescence marks the central cell outline (red line). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.013
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221737&req=5

fig5s2: The expression of neither constitutively-active ROP8 (CA-ROP8) nor dominant-negative ROP8 (DN-ROP8) under the control of the ROP8 promoter affect the F-actin structure (green) in the central cell.Autofluorescence marks the central cell outline (red line). Scale bar = 10 µm.DOI:http://dx.doi.org/10.7554/eLife.04501.013
Mentions: We observed that, upon entry into the central cell, the sperm cell nucleus becomes surrounded by an increasing number of F-actin cables (Figure 3A–D), followed by the formation of a dynamic aster-like structure around the sperm cell nucleus (Figure 3E–H). This aster-like structure of F-actin was only observed around the sperm cell nucleus in the central cell after plasmogamy (100%, n = 48) and was never observed in unfertilized central cells (0%, n = 72). Consistently, the time-lapse imaging showed the presence of aster-like F-actin structure only during sperm cell nucleus migration [100% (n = 5); 0% in unfertilized ovule (n = 14)].The F-actin aster-like structure migrated together with the sperm cell nucleus towards the central cell nucleus (Video 1), suggesting the involvement of specific actin regulators in this process. In plants, F-actin assembly is regulated by formins and ARP2/3 complex under the control of plant-specific Rho-GTPases (ROP; Craddock et al., 2012; Henty-Ridilla et al., 2013). Transcriptional profiling (Borges et al., 2008; Le et al., 2010; Wuest et al., 2010; Belmonte et al., 2013) suggested that ROP8 is specifically active in the central cell and the endosperm (Figure 5A). Indeed, we found that the ROP8 promoter is specifically active in the central cell (Figure 5B) and the fusion protein Venus-ROP8 associates to the plasma membrane of the central cell (Figure 5C). Consistently, the ectopic expression of GFP-ROP8 also showed its association to the plasma membrane in root epidermal cells (Figure 5—figure supplement 1), indicating that ROP8 sub-localization is controlled by general machinery common between the gametophytic central cell and somatic root epidermal cell. To understand the function of ROP8 in the central cell during fertilization, we investigated F-actin organization in central cells expressing constitutively active and dominant-negative forms (Yang, 2002) of ROP8 (CA-ROP8 and DN-ROP8, respectively) under the control of the ROP8 promoter. Although F-actin structures in both CA-ROP8 and DN-ROP8 appeared similar to WT (Figure 5—figure supplement 2), only DN-ROP8 lines showed that the sperm cell chromatin in the central cell remained condensed and unfused with the central cell nucleus, when the sperm cell chromatin had already fused with the egg cell nucleus and appeared decondensed (Figure 5D; Line 1, 42% defects [n = 98], Line 2, 36% defects [n = 102]). This phenotype was similar to the fertilization defect observed in central cells expressing DN-ACTIN (Figure 4).10.7554/eLife.04501.011Figure 5.ROP8 is specific to the central cell and involved in 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.