The Systems Biology of the Circadian Control of Freezing Tolerance in Arabidopsis thaliana

Abstract

The Arabidopsis thaliana circadian clock is involved in regulating several plant systems including light signalling, germination and the cold signalling pathway. The role of the circadian clock in regulating far-red and red light induced dormancy and germination, however, is not well understood. In this thesis it is shown that the circadian clock does not seem to be involved in regulating far-red light induction of dormancy, but that the TIMING OF CAB EXPRESSION 1 (TOC1) gene is vital for red light induced germination to occur. In Arabidopsis thaliana, the transcription factors, C-REPEAT BINDING FACTORs (CBFs) are key components of the cold acclimation pathway. The expression of the CBFs has recently been shown to be regulated by the circadian clock; however, our understanding of how the CBFs are regulated by the clock is far from complete. In the main focus of this thesis a systems biology approach was utilised to try and better understand the circadian regulation of plant cold responses, specifically the manner by which the circadian clock regulates the cold acclimation pathway C-REPEAT BINDING FACTOR 3 (CBF3) gene. Freezing tolerance assays were carried out to increase our knowledge of the clock regulation of the cold signalling pathway. Circadian clock mutant lines without previously reported freezing tolerance phenotypes were identified in the TOC1 mutant, toc1-101, and the EARLY FLOWERING 3 (ELF3) and LUX ARRHYTHMO (LUX) mutants elf3-1 and lux. The freezing assay data was used to influence model designs for the circadian regulation of CBF3 expression. Several potential models of CBF3 regulation were created. The models were then optimised against publically available microarray gene expression data. Model selection using a Corrected Akaike Information Criterion (AICc) was utilised to establish models that best fit biological data. Predictions made by the models were then tested, thus leading to the establishment of new circadian clock mechanisms of CBF3 being discovered. 4 The modelling procedure predicted the involvement of the Evening Complex (EC) and TOC1 in regulating CBF3 expression as well as the already reported regulation by LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1); the PSEUDO-RESPONSE REGULATORS (PRRs) which had been predicted as direct regulators of the CBFs were not needed to produce correct CBF3 expression in any of the potential models. The direct TOC1 and Evening Complex regulation of CBF3 promotion was then confirmed by chromatin immunoprecipitation.