Author ORCID Identifier

https://orcid.org/0000-0002-5908-5868

Date Available

5-28-2023

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant and Soil Sciences

First Advisor

Dr. Tomokazu Kawashima

Abstract

Flowering plants have evolved a unique double-fertilization process. Two sperm cells fuse with two female gametophytic cells, the egg and central cells within the ovule, giving rise to the embryo and endosperm, respectively. Sperm cells in flowering plants are nonmotile and delivered in close proximity to the egg and central cells by the pollen tube. Flowering plants have established filamentous actin (F-actin) based sperm nuclear migration system for successful fertilization. Prior to fertilization, the female gamete forms a mesh-like structure of F-actin that shows constant inward movement from the plasma membrane periphery to the center of the cell where the female gamete nucleus resides. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for sperm nuclear migration 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 in Arabidopsis thaliana. We demonstrate that an ARP2/3-independent WAVE/SCAR-signaling pathway and actin nucleator formins regulate F-actin dynamics in the central cell for fertilization. We also identify that myosin class 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 sperm nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

After double fertilization, the central cell develops into endosperm and in flowering plants it undergoes repeated mitotic nuclear divisions without cytokinesis, resulting in a large coenocytic endosperm that then cellularizes. Growth during the coenocytic phase is strongly associated with the final seed size; however, a detailed description of the cellular dynamics controlling the unique coenocytic development in flowering plants has remained elusive. By integrating confocal microscopy live-cell imaging and genetics, we have characterized the entire development of the coenocytic endosperm of Arabidopsis thaliana including nuclear divisions, their timing intervals, nuclear movement, and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive F-actin organization. Microtubules promote nuclear movement after division while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and mature seed. Characterizing cytoskeleton dynamics in real-time throughout the coenocyte endosperm period provides foundational knowledge of plant coenocytic endosperm development.

Furthermore, in another project, we investigated the importance of the initial stage of seed development (the lag phase), on final seed size determination in soybean (Glycine max L. Merr.). We investigated soybean seed phenotypes grown in a greenhouse using different source-sink manipulations (shading and removal of flowers and pods) during the lag phase. We show that assimilate supply is the key factor controlling flower and pod abortion and that the assimilate supply during the lag phase affects the subsequent potential seed growth rate during the seed filling phase. Our results provide insight into the mechanisms whereby the lag phase is crucial for seed development and final seed size potential, essential parameters that determine yield.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.384

Additional Files
Supplementary information.zip (74845 kB)
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