ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants

Abstract

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration inArabidopsis thaliana(Arabidopsis) andNicotiana tabacum(tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in theArabidopsiscentral cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

An F-Actin Mega-Cable Is Associated With the Migration of the Sperm Nucleus During the Fertilization of the Polarity-Inverted Central Cell of Agave inaequidens

“…Recent analysis of central cell fertilization in A. thaliana shows that an F-actin mesh-like structure which moves from the periphery to the center of the cell, along with an F-actin star-shaped structure that encloses the sperm nucleus, mediates the transit of the latter to the central cell nucleus ( Kawashima et al, 2014 ). Similar processes have been observed in other plant models ( Ohnishi and Okamoto, 2017 ) and the mechanism has been proposed as a general one for Angiosperms ( Ali et al, 2020 ). However, none of the plant species analyzed thus far have possessed a chalazal-polarized central cell nucleus.…”

“…While in Arabidopsis central cell F-actin forms a mesh-like structure growing from and attached to the plasma membrane by formins and ROP8, Agave actin-tunnel is predominantly formed by parallel actin cables that run along the chalazal-micropylar axis from the central cell nucleus ( Figure 3C and Supplementary Video 1 ). When Ali et al (2020) inhibited the myosin activity with 50 mM BDM (2, 3-butanedione monoxime) in the Arabidopsis central cell, they observed the straightening of the F-actin meshwork, which adopted a similar configuration to those observed in Agave .…”

“… Kawashima et al (2014) demonstrated that F-actin, but not microtubules, transports the immotile sperm nuclei during Arabidopsis fertilization. Once inside the central cell, the second sperm nucleus is surrounded by an aster-shaped F-actin structure ( Kawashima et al, 2014 ) that migrates in synchrony with inward moving (plasma membrane to central cell nucleus) F-actin cables attached to the plasma membrane by formins and ROP8 ( Ali et al, 2020 ; Ali and Kawashima, 2021 ). These F-actin cables form a mesh-like structure whose movement and stability depend on class XI myosin XI-G ( Ali et al, 2020 ).…”

“…Once inside the central cell, the second sperm nucleus is surrounded by an aster-shaped F-actin structure ( Kawashima et al, 2014 ) that migrates in synchrony with inward moving (plasma membrane to central cell nucleus) F-actin cables attached to the plasma membrane by formins and ROP8 ( Ali et al, 2020 ; Ali and Kawashima, 2021 ). These F-actin cables form a mesh-like structure whose movement and stability depend on class XI myosin XI-G ( Ali et al, 2020 ). A similar F-actin arrangement and meshwork movement have been observed in rice zygotes ( Ohnishi and Okamoto, 2017 ), which suggested the migration mechanism of this sperm nucleus might be general among angiosperms ( Ali et al, 2020 ).…”

“…These F-actin cables form a mesh-like structure whose movement and stability depend on class XI myosin XI-G ( Ali et al, 2020 ). A similar F-actin arrangement and meshwork movement have been observed in rice zygotes ( Ohnishi and Okamoto, 2017 ), which suggested the migration mechanism of this sperm nucleus might be general among angiosperms ( Ali et al, 2020 ). Nevertheless, a physical connection between the aster-shaped structure and the inward moving F-actin cables has not been established yet.…”