The Role and Position of Diel [Ca2+]cyt Oscillations in the Arabidopsis thaliana Circadian Clock

Abstract

Cytosolic free calcium (Ca2+cyt) is a ubiquitous second messenger in eukaryotes. In Arabidopsis thaliana, diurnal or circadian (diel) rhythms in [Ca2+]cyt have been widely documented. There is evidence to suggest that these diel [Ca2+]cyt oscillations modulate different signalling pathways, including photoperiodic signal transduction, gating responses to endogenous and environmental stimuli and feed-back entrainment of the core circadian clock itself. However, direct evidence for the role of Ca2+ in clock entrainment or as an output from the clock is lacking, and the question of the functional role of diel [Ca2+]cyt oscillations remains open. The role of diel [Ca2+]cyt rhythms in A. thaliana and their relationship relative to the central molecular oscillator was investigated. While it was found that diel [Ca2+]cyt oscillations persist throughout the life cycle of A. thaliana, I found no indication that diel [Ca2+]cyt rhythms are involved in photoperiodic signalling. Furthermore, I demonstrated that normal diel [Ca2+]cyt oscillations persist even in the absence of a functioning core circadian clock, indicating that, contrary to the accepted view, diel [Ca2+]cyt oscillations are not directly controlled by the core circadian clock, but are more probably generated by a non-transcriptional oscillator. In silico analysis of the amino-acid sequences of the 12 core clock proteins revealed that TOC1 contains a putative EF-hand and may therefore provide a route into the molecular oscillator for diel [Ca2+]cyt signals. The TOC1 sequence was altered to eliminate the Ca2+ coordinating residues but attempts to express this protein in E. coli, N. benthamiana and Baculovirus were unsuccessful. Complementation of the A. thaliana toc1-1 mutant with transgenes containing the endogenous TOC1 promoter sequence upstream of the wild type or the altered TOC1 sequences were also unsuccessful. A series of experiments were conducted to provide empirical data for Boolean Logic models of circadian rhythmicity that would enable further characterisation of the potential link between diel [Ca2+]cyt oscillations and TOC1.