IDENTIFICATION AND CHARACTERIZATION OF NOVEL TARGETS FOR A SUBFAMILY OF ARABIDOPSIS CALMODULIN-LIKE (CML) PROTEINS

Abstract

Calcium is a ubiquitous second messenger in signal transduction pathways of eukaryotic cells. In plants, calcium signalling plays critical roles in a variety of developmental processes as well as during cellular responses to abiotic and biotic stress. The evolutionarily conserved calcium sensor, calmodulin (CaM), responds to calcium signals and, through protein-protein interactions, facilitates downstream responses. Plants possess an additional and unique family of “calmodulin-like” (CML) calcium sensors which, in Arabidopsis thaliana, contains 50 members. Despite this expanded complexity, few of the binding partners and/or in vivo functions of CMLs are known. Here, utilizing a Y2H screen, I demonstrate the interaction of CaM and a subfamily of biochemically distinct CMLs, CML13 and CML14, with three protein families in Arabidopsis which possess a conserved CaM binding motif. Through in planta split-luciferase and relocalization assays, my data suggests that at least select members of these families are capable of interacting with CaM, CML13, and CML14 in planta. Additionally, delineation through the Y2H system suggests that multiple binding motifs in tandem are able to interact with CaM/CMLs with a greater affinity than single motifs. This data is further supported by advanced biophysical approaches which show increased binding affinities when multiple binding motifs are present but also revealed the complex binding rearrangements that occur with these motifs. Finally, in an attempt to reveal potential in vivo functions of CML13 and CML14, numerous overexpression and silencing transgenic Arabidopsis tools were generated, and revealed major morphological alterations when CML14 was suppressed. Collectively, my data suggests novel functions for plant CMLs in gene regulation and cytoskeletal activity.