Aoki, Megan Marie

Cytokinins (CKs) are a pervasive group of growth-promoting signaling molecules spanning every kingdom of life. Their roles are best known in plants, where they act as phytohormones controlling nearly all aspects of plant growth and development. CKs continue to be detected in new organisms, posing questions about their roles in such widespread forms of life. The research presented in this thesis, therefore, investigated CK dynamics in a non-plant system using the simple eukaryotic model, Dictyostelium discoideum. Prior to this thesis, CKs were established as key intercellular signals necessary for proper development of D. discoideum – specifically in the induction of sporulation and maintenance of spore dormancy. However, there were no documented roles of CKs prior to the late stages of multicellular development. Comprehensive mass spectrometric screening for CKs detected six novel CK forms during all stages of D. discoideum growth and development. Based on these findings, a model was proposed that mapped CK biosynthesis in D. discoideum. The CK profiles indicate that there are differing dominant CK forms during vegetative growth and early development compared to those detected during late multicellular development. This led to the hypothesis that CKs have different roles during the distinctive life cycle stages of D. discoideum. This hypothesis was tested by generating knockout and overexpression strains of the key, primary CK biosynthesis gene, iptA, to investigate potential expanded roles for CKs during growth and the early stages of D. discoideum development. iptA-deficiency resulted in cytokinesis defects and both iptA-deficiency and overexpression caused altered mitochondrial morphology, dysregulated TCA cycle and amino acid metabolism, as well as increased levels of the energy metabolite, AMP. These combined phenotypes were suggestive of mitochondrial-associated dysfunction in vegetative growth and provided the first evidence of expanded roles of CKs during the D. discoideum life cycle. This was the first metabolomics-based evidence of CKs influencing mitochondrial function in D. discoideum. Lastly, a key CK-activating enzyme was functionally characterized, DdLOG, and additional CK biosynthesis enzymes were identified for future examination. Together, the findings of this thesis provide insights into: CK biosynthesis in a non-plant associated model; new roles for CKs during the D. discoideum life cycle; and CK interactions with mitochondria. The methods established as part of this thesis can be used as a foundation for characterizing further CK biosynthesis enzymes and as a guide for detecting subtle sub-cellular phenotypes related to CK metabolism in D. discoideum and other CK-producing organisms.

Author Keywords: cytokinin biosynthesis, cytokinins, Dictyostelium discoideum, IptA, mass spectrometry, mitochondria