Abstract

CML23 and CML24 are potential Ca2+ sensors that are involved in the regulation of the Arabidopsis thaliana circadian clock. These plant-specific family of Ca2+ sensor proteins may have overlapping roles in regulating the Arabidopsis central circadian oscillator. A previous study proposed that transcription of core oscillator genes are affected in the cml23-2cml24-4 double mutant. However, how the circadian oscillator is regulated by CML23 and CML24 remains to be explained because CML23 and CML24 are not thought to be transcription factors. It is likely that CML23 and CML24 regulate the circadian oscillator through interacting intermediaries. Conformational changes undergone by CML23 and CML24 , as a consequence of Ca2+ binding to the 4 EF hands, may facilitate the recognition of target proteins. In this study, co-immunoprecipitation, epistatic analysis and subcellular localization are used to characterize the function of CML23 and CML24 in the regulation of the Arabidopsis thaliana circadian clock. To identify the putative interacting partners of CML23 and CML24 , Myc-tagged CML23 and FLAG -tagged CML24 constructs were made and introduced into the Col-0 ecotype of Arabidopsis. In vivo co-imunoprecipitation of the transgenic lines will now be carried out. Database queries from www.ATTED-II have resulted in identification of CML5 as a gene that is highly co-expressed with CML24 . Circadian leaf movement rhythm of the cml5 mutant is being assessed. In addition, study of the epistatic relationship between core oscillator genes and cml23-2cml24-4 may indicate potential points of entry for CML23 and CML24 into the circadian system. The known clock loss of function mutants, cca1-11, lhy-21, gi-11, elf3-4, elf4-1, toc1-2, ztl-3 and lux-4 were crossed with cml23-2cml24-4 mutants. Circadian leaf movement rhythm analysis will be used to screen for the phenotype of the triple mutants. We also hypothesize that CML23 and CML24 are cytosolic proteins, which play a role in signal transduction from the cytosol into the nucleus. RFP -tagged CML23 and GFP -tagged CML24 transgenic plants that we have made will be analysed to identify the subcellular localization of CML23 and CML24 .