The Arabidopsis Calmodulin-Like Protein, CML39, Regulates Early Seedling Development and Fruit Formation

Abstract

Calcium (Ca2+) is one of the most ubiquitous and versatile second messengers in eukaryotes. In the Ca2+ signaling paradigm, cytosolic Ca2+ oscillations, evoked by environmental stimuli, are detected by Ca2+-binding proteins, termed Ca2+ sensors, that help coordinate physiological responses by binding to and regulating the activities of various other proteins. Calmodulin (CaM) is an evolutionarily-conserved eukaryotic Ca2+ sensor involved in many signal transduction pathways. Intriguingly, plants also possess large families of unique Ca2+sensors related to CaM and known as calmodulin-like (CML) proteins. Several CMLs have been implicated in developmental and stress-response signaling but the roles of most CMLs remain unknown. Among these CMLs, CML39 is of particular interest due to its roles in early seedling establishment. The focus of my thesis was to determine whether CML39 played additional roles in plant development and to also characterize the mechanism(s) underlying CML39-mediated physiological phenomena. Thus, I employed a multi-disciplinary approach to address this question. Detailed analysis of cml39 T-DNA insertional knockout plants under various developmental stages identified several abnormalities in these mutants. Relative to wild-type (WT), cml39 displayed aberrant germination behaviour, reduced hormone sensitivity in seeds, altered seed coat characteristics, reduced fertility and de-repression of seed maturation genes in seedlings. The pleiotropic phenotype of cml39 mutants confirms that CML39 regulates diverse aspects of plant development. RNAseq analysis of cml39 seedlings revealed broad perturbations in global gene expression and provided some insight into CML39-regulated pathways. In addition, two trihelix transcription factors, Arabidopsis 6b-interacting 1-like (ASIL) -1 and -2, were found to interact with CML39 in yeast 2-hybrid and in planta split-luciferase protein-protein interaction analysis, suggesting that CML39 may regulate gene transcription in some tissues. Genetic analysis of cml39 and asil mutants indicated that CML39-ASIL interaction may be responsible for dormancy maintenance in developing seeds and repression of seed maturation program in vegetative tissue. Collectively, the findings presented in this thesis suggest that CML39 plays an integral role in regulation of seed- and fruit development, dormancy induction and maintenance, hormone sensitivity of seeds, and transition from seed to seedling phase, thus emphasizing the significance of this Ca2+ sensor in plant development.