Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Plant Biology
First Advisor
Mary Tierney
Abstract
Perhaps the most striking difference between eukaryotic and prokaryotic life is the compartmentalization of essential biochemical functions to membrane-bound organelles within eukaryotic cells and the general lack of such structures in prokaryotes. This strategy requires several challenges to be addressed including the population and maintenance of distinct suites of proteins within each organelle. To meet this challenge, a network of membrane-bound vesicles and organelles collectively known as the endomembrane system is used to direct integral membrane proteins and intraluminal cargo to their sites of action. Major components of the endomembrane system include the nucleus, endoplasmic reticulum, Golgi-apparatus, trans-Golgi network, plasma membrane, early endosomes, multi-vesicular bodies, and the vacuole. The proteins within plant plasma membranes enable efficient resource utilization and responses to environmental changes, making their dynamic regulation of great interest. Substantial progress has been made in understanding several key trafficking pathways in plants that converge at the plasma membrane, including the conventional secretory pathway and the multi-vesicular body-mediated degradation of endocytosed membrane proteins. Investigations into these processes are aided by the well conserved nature of these pathways between yeast, mammals, and plants. Accordingly, when key regulators of protein trafficking are described in one system, the identification of homologous proteins through sequence identity or structural homology can provide the basis for the expansion of new discoveries across systems. Recently, while our lab was studying the role of VPS26C in root hair growth in Arabidopsis thaliana, a homologous protein in mammalian cells was described as a central member of a complex involved in the recycling of endocytosed plasma membrane proteins back to the plasma membrane. The complex was named retriever by McNally et al. (2017), and we were able to demonstrate that a similar complex consisting of VPS35A-VPS29-VPS26C is formed in Arabidopsis. Furthermore, the human ortholog of VPS26C was able to complement the short root hair phenotype of vps26c-1 mutants, suggesting the conservation of function between these proteins. The work in this dissertation builds on these discoveries by investigating two proteins, CCDC22 and CCDC93, that are important regulators of retriever in mammalian cells and encoded within the genome of Arabidopsis. Through complementation assays we demonstrate roles for CCDC22 and CCDC93 in the growth of roots and root hairs. These observations and the realization that both vps26c-1 and ccdc93-1 knockout mutations can suppress the short root hair phenotype of plants defective for a vacuolar SNARE, vti13, suggest CCDC22, CCDC93, and VPS26C participate in a shared root hair developmental pathway. Double mutants ccdc22-1ccdc93-1 were generated by crossing and CRISPR/Cas9 induced mutagenesis was used to generate ccdc22-3vps26c-1 double mutants. Analysis of the root and root hair phenotypes of these double mutants does not suggest additivity, further indicating that these genes are common to the same developmental pathways. Finally, fluorescence microscopy and pharmacological approaches were used to investigate plasma membrane trafficking in these mutant backgrounds and showed the double mutants to have altered trafficking capabilities compared to wild-type plants, suggesting a conserved role in plasma membrane trafficking.
Language
en
Number of Pages
133 p.
Recommended Citation
Lewis, Connor, "Genetic And Functional Characterization Of Ccdc22 And Ccdc93 In Arabidopsis Root And Root Hair Growth" (2025). Graduate College Dissertations and Theses. 2030.
https://scholarworks.uvm.edu/graddis/2030
