Natural variation in the Arabidopsis thaliana circadian clock as a determinant of flowering time: a quantitative genetics and genomics study

The circadian clock is an endogenous mechanism present in plants that enables anticipation of upcoming environmental changes. In this way, the clock thus facilitates the correct timing of physiological and developmental events. An internal clock synchronized with the external environment ensures that plants flower under favorable environmental conditions, and thus, provides fitness advantages under natural habitats. In Arabidopsis, the molecular framework of the circadian-clock machinery is constituted by a network of transcription/translation-based feedback loops. The main loop that comprises CCA1/LHY and TOC1, is connected with the morning loop comprising PPR7/PRR9 through CCA1/LHY. TOC1 establishes a link of this main loop with an as of yet unknown component (Y) in the evening loop. The flowering-time component GI fulfills some of the requirement of ‘Y’. However, functional studies do not fully support the GI to TOC1 link. ELF3 is another clock component whose function is not fully understood. In the past, efforts for functional placement of ELF3 were mainly hampered by the arrhythmic nature of available elf3 alleles. In chapter 3 of this thesis, I described the characterization of a natural allele of ELF3 (ELF3-Sha) that displays conditional rhythmicity and provides evidences of the ELF3 mode-of-action in the clock. From this, in chapter 4, using quantitative genetics and genomics studies in Arabidopsis, I identified the possible positions of the new allelic variants that connect the speed of circadian oscillation to flowering time. Adaptation of Arabidopsis accessions to varying environmental conditions of light and temperature at different geographical areas has provided a force for the selection of allelic variants within the circadian clock. Previously, the location of such allelic variants affecting speed of the clock was identified in a Bay x Sha recombinant inbred line (RIL) population. This population was modified with a CCR2::LUC promoter-reporter system, which provides robust estimates of the clock activity, and hence, the speed of the clock can be accurately measured. In such experiments, a major-effect periodicity QTL was detected, where the presence of Sha allele at the locus caused a short-period phenotype. I report in chapter 3 the positional isolation of this QTL and revealed that ELF3 was the underlying gene. ELF3 has been reported to be a nuclear-localized protein required for both the generation of circadian rhythms and light input to the circadian clock. I found that the casual polymorphism in the ELF3-Sha allele was an encoded replacement in highly conserved amino acid that caused the short-period phenotype. Interestingly, this phenotype was light dependant. In constant darkness, a different phenotype was found, and this was a reduction in clock function with a movement towards arrhythmic oscillations. The cellular basis of the ELF3-Sha protein defect correlated with an increase in cytosolic distribution. Taken together, my data suggests that the light and circadian action of ELF3 in the clock is dependent on nuclear recruitment to initiate repressive action in the clock network. Characterization of ELF3-Sha established the notion that natural variation in Arabidopsis accessions provides an extraordinary resource to study complex physiological mechanisms. Therefore, I decided to exploit this natural resource to investigate the relationship between the circadian clock and flowering time, which is reported in chapter 4. For that, I selected three Arabidopsis accessions from geographically diverged areas: two from northern latitudes and one from the equator. Whole genome re-sequencing of these accessions revealed that, on a genome-wide scale, the equatorial accession is highly diverged compare to the northern accessions. This encouraged me to utilize these accessions in quantitative-genetic studies. Pair-wise crosses were made to generate RIL sets that were screened for circadian periodicity and flowering-time traits. Construction of genetic maps followed by QTL mapping revealed several loci controlling the speed of clock and flowering time. Many of these QTLs were localized with the known genes controlling both of these traits. Novel loci were also detected. Validation of a major QTL in a near isogenic line, followed by sequence comparison of the candidate gene in parental accessions, revealed that a single encoded amino-acid change in GI potentially causes the periodicity acceleration. Further, identification of flowering-time loci overlapping with the position of known clock genes provided the mechanistic explanation of the relationship of clock with flowering time. Taken together, my research strongly advocates the importance of the natural-variation studies in understanding intricate physiological pathways and their interactions with each other.