Herrero Serrano, Eva (2011) A molecular basis of ELF3 action in the Arabidopsis circadian clock. PhD thesis, Universität zu Köln.
The circadian clock anticipates daily environmental changes and optimizes timing of physiological events. Circadian systems are present in most living organisms. In Arabidopsis, circadian components are arranged in positive/negative regulatory feed-back loops. The core loop is arranged by the morning transcription factors LHY and CCA1, and the evening pseudo-response regulator TOC1. The morning loop reciprocally connects LHY and CCA1 to the TOC1-sequence-related components PRR9 and PRR7. Other genes, when mutated, display a clock phenotype, but not all of such genes have been placed into the clock model. For instance, three evening genes, ELF3, ELF4, and LUX, have been found to be essential for circadian function. Clock- and light-signaling networks are tightly interconnected. Light input to the clock is mediated by photoreceptors, such as the phytochromes. ELF3 and ELF4 play a pivotal role in the generation of circadian rhythms and in the integration of light signal to the clock mechanism. Both encoded proteins are reported to be located in the nucleus, and both the elf3 and the elf4 mutants display a similarly arrested clock. In this thesis, ELF3 was found to be genetically downstream of ELF4 within the same clock signaling pathway required to sustain circadian rhythms. Moreover, I found that ELF3 and ELF4 proteins physically interact. This interaction correlated with an increase of ELF3 nuclear localization. These observations are consistent with a role of ELF4 as an effector that promotes ELF3 activity to lengthen circadian periodicity. A functional complementation approach identified three functional modules in the ELF3 encoded protein. The N-terminus and middle domains mediate interaction with phyB and ELF4, respectively. The C-terminus domain was found to be required for ELF3 nuclear localization. Thus, ELF3 is a multifunctional protein that interacts with both light-signaling and clock components. The molecular function of ELF3 had previously remained elusive. PRR9 expression was found to be down-regulated in ELF3 and ELF4 over-expressors. Interestingly, I found that ELF3 physically associated with the same conserved region in the PRR9 promoter as the transcription factor LUX. I found that LUX was genetically downstream of ELF4, and that LUX required ELF3. Taken together, I proposed that ELF3, ELF4, and LUX are part of an evening-clock complex required to repress PRR9 expression, and to sustain circadian oscillations. ELF3 has been reported to be crucial to buffer light input to the oscillator. Photoreceptors and ELF3 play an opposite role in light-mediated acceleration of circadian periodicity, where photoreceptors shortens, and ELF3 lengthens, circadian period under constant light. Interestingly, I found that the N-terminus of ELF3 was not essential for ELF3 circadian function, but that mediated the physical interaction of ELF3 to phyB. An elf3 complementation line deleted for its N-terminus displayed hyposensitivity to the period-shortening effect induced by constant-red light. Therefore, I hypothesized that phyB interaction to the N-terminus of ELF3 mediates light-repression of ELF3 action in circadian-periodicity. In chapter 4, further characterization of the weak allele elf3-12 supported the role of ELF3 as a decelerator of circadian periodicity. The elf3-12 mutation encodes an amino-acid replacement in a conserved box within the ELF4-binding domain. The elf3 12 coding region led to robust expression of ELF3-12 protein, and ELF3-12 retained the capacity to bind both ELF4 and phyB. elf3-12 displayed light-dependent short-period phenotype that was enhanced by phytochrome over-expression. Moreover, elf3-12 displayed hypersensitive to red-light-resetting pulses. Thus, I found that elf3-12 is attenuated in its function to repress light input to the clock and/or and increased phy-mediated repression of ELF3 function. elf3-12 was the first described elf3 weak allele. My characterization of a collection of elf3 TILLING alleles led to the identification of novel short- and long-period alleles that I predict will expand current understanding of the role of ELF3 as an integrator of light signals and as a core-clock component. Taken together, my thesis has placed ELF3 within the circadian mechanism. ELF3, ELF4, and LUX are part of an evening-repressor complex required to sustain circadian function. The genetic interaction of these three genes is consistent with a hierarchy of complex assembly. In this, I propose that ELF4 works as an effector protein that activates ELF3, possibly by increasing the ELF3 nuclear pool. Then, the association of both ELF3 and LUX to the PRR9 promoter is required for transcriptional repression of PRR9. Additionally, I propose that ELF3 function in circadian periodicity is modulated by its interaction partners by a competition between a positive effect of ELF4 and a light-mediated-negative effect of phyB. This is consistent with ELF3 being a multifunctional protein that integrates light signals as a core-oscillator component.
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