Genetic Analysis of two Evening Genes in the Arabidopsis thaliana Circadian Clock

The circadian clock generates biological rhythms that have an approximate period-length of 24 h. This endogenous timing mechanism is integrated in many signaling pathways and facilitates an optimal phase-relationship between internal and external rhythms. For example, the expression of photosynthesis genes is reflected in a rhythm that anticipates the environmental light-dark cycle. The circadian-clock system in plants has been investigated at the molecular level, and similar to other lineages, the plant clock has been found to consist of transcription/translation feedback loops. Currently, the circadian network is described to contain three interlocked feedback loops, and the CCA1/LHY-TOC1 loop is core within this feedback network. CCA1 and LHY are two MYB transcription factors that function in the morning and repress the expression of TOC1. Accordingly, TOC1 expression is delayed and peaks in the evening, and TOC1 function leads to subsequent induction of CCA1/LHY expression in the end of the night. How the rest of the components of the circadian system are connected to the property of the central oscillator remains unclear. In this thesis, two components of the circadian system, ELF4 and ELF3, were investigated. Both ELF4 and ELF3 are believed to function as inputs to the CCA1/LHY-TOC1 loop and their loss-of-function leads to arrhythmic behavior of the clock. ELF4 and ELF3 are unrelated in sequence, and both genes contain no evolutionarily conserved domains with known function. To this end, reverse-genetic approaches were applied in order to characterize the structure-function relationship of the ELF4- and ELF3-encoded products. In addition, the ELF4 and ELF3 circadian activities were related to each other, and both sequences were characterized phylogenetically. The comparison of members in the plant-specific ELF4 family revealed that two major subclades (ELF4 and EFL) are present in the phylogeny. The ELF4 consensus consists of a single domain, which is predicted to fold into a conserved alpha-helical structure. Accordingly, it was hypothesized that changes in this structure would correlate with ELF4 function. This was proven in two ways. First, ELF4-related sequences outside the ELF4 subclade were insufficient in complementation of the arrhythmic phenotype of the elf4 null mutant. Second, the effects of point mutations affecting ELF4 structural conservation correlate with the severity of the mutant phenotype. In addition, analysis of efl circadian phenotypes suggests roles for the EFL genes in fine-tuning of the circadian oscillator. The analysis of the elf4 point mutations concluded that ELF4 function is tightly connected to the light-induced expression of CCA1. This result confirmed previous findings for ELF4, and the relationship between ELF4 and the CCA1/LHY-TOC1 loop was further investigated by analyzing the effects of ELF4-overexpression on circadian parameters; this was compared to new studies on the elf4 loss-of-function mutation. It was found that there is a doseeffect of ELF4 on clock period, both at the molecular level and in plant physiological performance. Additionally, rhythmic expression of ELF4 is not required for sustained clock activity under constant conditions, and ELF4 has a critical role in clock entrainment to the light- Zeitgeber, possibly via a gating function. Epistatis analysis of ELF4 and ELF3 defined ELF3 as the most upstream regulator of light input to the central oscillator. ELF3 function was further analyzed by characterization of a newly found allele termed elf3-G12. This mutant was found to display subtle and clock-specific phenotypes. Expression analyses revealed that ELF3 misexpression confers phase shift of central clock genes and ELF3 is likely to be most associated with TOC1 function under free-running conditions. Furthermore, it was determined that the phenotype of the elf3-G12 mutant is related to PHYB-interaction, supporting the position of ELF3 at the convergence point of light transduction and input to the circadian clock