The regulation and function of CML23 and CML24: Arabidopsis thaliana genes encoding calcium-binding proteins implicated in abscisic acid response, floral transition, and ion homeostasis
Delk, Nikki Ayanna
Doctor of Philosophy
Despite their complacent appearance, plants are highly perceptive of stimuli in their environment. Responses to these stimuli enable plants to acclimate to the conditions of their local environment. Fluctuations in cellular calcium (Ca2+) have been implicated as critical signals for transducing external stimuli into intracellular information. How Ca 2+ signals are decoded in plants is largely unknown. The full Arabidopsis genome sequence enabled the identification of a family of 50 genes encoding potential Ca2+ sensors that are related to the quintessential Ca2+-binding protein, calmodulin (CaM). These C&barbelow;aM&barbelow;-l&barbelow;ike or CML proteins have the potential to mediate Ca2+ signaling in plant cells. My thesis work is focused on elucidating the potential physiological significance of two related CML proteins, CML23 and CML24 (also known as TCH2). CML23 and CML24 share over 40% amino acid sequence identity with CaM and have 4 EF-hand Ca2+-binding motifs. CML24 is likely a Ca 2+ sensor because it can undergo Ca2+-dependent changes in hydrophobic interaction chromatography and migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. These characteristics may enable CML24 to interact with, and potentially regulate, targets in a Ca2+-influenced manner. Reverse transcriptase polymerase chain reaction (RT-PCR) and reporter transgenic analysis indicate that CML23 and CML24 expression occurs in all major organs. Quantitative RT-PCR (QRT-PCR) reveals CML24 transcript levels are increased from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid (auxin). In addition, CML24 over- and underexpressing transgenics, cml24 point mutants, and cml23/cml24 double mutants have phenotypes which demonstrate these proteins play roles in flowering time regulation, ion sensitivity, and ABA inhibition of germination and seedling growth. Phenotype analysis also suggests CML23 and CML24 may have a role in de-etiolation (light-induced inhibition of hypocotyl elongation), chlorophyll accumulation, and response to pathogens. Furthermore, a yeast two-hybrid screen suggests CML24 interacts with an autophagy protein. Elucidating CML23 and CML24 expression patterns and physiological functions has provided additional insight into the roles of Ca2+ and Ca2+-binding proteins in plant development and response to the environment.
Molecular biology; Plant physiology