Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Program Name/Specialization

Biological and Chemical Sciences

Faculty/School

Faculty of Science

First Advisor

Matthew Smith

Advisor Role

Supervision, Collaboration on Experimental Design, Grant Funding, Feedback on Dissertation

Second Advisor

Masoud Jelokhani-Niaraki

Advisor Role

Co-Supervision, Collaboration on Experimental Design, Grant Funding, Feedback on Dissertation

Abstract

Global agriculture and food security face challenges from climate change, population growth, and other human-driven pressures. Enhancing our understanding of plant biology, particularly chloroplast function, is critical for improving crop yield and stress resilience. As the sites of photosynthesis and key regulators of plant responses to environmental cues, chloroplasts depend on the precise import of thousands of nuclear-encoded proteins to maintain their biogenesis and function. While much is known about the core translocon components in the chloroplast outer envelope membrane that form the TOC complex, the mechanisms underlying their targeting, assembly, and regulation remain incompletely understood.

Plastids, including chloroplasts, evolved from an ancient cyanobacterial endosymbiont and now rely on sophisticated protein targeting pathways to import nuclear-encoded proteins. The chloroplast outer envelope membrane proteome, in particular, remains relatively unexplored. Through a bioinformatic approach, the work in this dissertation expanded the known list of chloroplast outer envelope membrane proteins in Arabidopsis thaliana from 117 to 138 and highlighted novel signals and pathways in protein targeting for this proteome that warrant further investigation. This includes the C-terminal “reverse transit peptide-like sequences” of TOC159 receptors.

Focusing on TOC159 receptors, a core component of the TOC complex, this dissertation investigated the mechanisms guiding their targeting, membrane integration and assembly into maturing TOC complexes. Targeting assays in A. thaliana protoplasts revealed that TOC159 receptors use a novel bipartite targeting signal at their C-terminus composed of a β-signal (G-Q-Φ-[ST]-Φ-[RK]-X-[SN]-[ST]) essential for targeting, and a transit peptide-like sequence that enhances targeting efficiency. Biophysical approaches demonstrated that this transit peptide-like sequence interacts preferentially with chloroplast-specific galactolipids, suggesting lipid-mediated targeting specificity.

Finally, using bimolecular fluorescence complementation, it was demonstrated that while GTPase domains of TOC33 and TOC159 receptor homologs can dimerize indiscriminately, the presence of TOC159 receptor acidic domains restricts certain interactions. These findings support a model in which the acidic domains drive the assembly of distinct TOC complexes that form photosynthetic and non-photosynthetic import pathways. Overall, this work deepens our mechanistic understanding of intracellular protein trafficking to the chloroplast outer envelope membrane of plant cells and informs future efforts to better understand TOC complex architecture and function toward engineering plant productivity and resilience through targeted manipulation of plastid biogenesis.

Convocation Year

2025

Convocation Season

Fall

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