Quantitative genetic analysis of temperature entrainment in the Arabidopsis thaliana circadian clock

The circadian clock is an internal mechanism that measures external time and generates overt rhythms. About 90% of the transcripts in the Arabidopsis thaliana genome are rhythmically expressed (Michael et al., 2008). Thus, all cellular process can be clock controlled to generate rhythmic physiological responses. Mathematical modeling predicted that the basic clock framework that generates various rhythmic outputs is comprised of three interlocking-feedback loops. The 24-hour rhythms are generated by the main CCA1/LHY-TOC1 feedback loop (Alabadi et al., 2001). Other genes, such as PRR7/PRR9 and GI, were shown to participate in morning or evening loops to fine tune rhythmicity (Locke et al., 2006; Zeilinger et al., 2006). This model takes in account experimental data generated under light-dark cycles. From this model, we can now hope to add environmental inputs, such as light and temperature entrainment, as integrated mechanisms within the circadian oscillator. What is relevant here is that in addition to light, temperature can also entrain the circadian oscillator. Whereas some understandings of light effects are known, it remains unclear how temperature sets the plant-circadian clock. In this thesis, I investigated temperature entrainment, as compared to light-dark entrainment, on the Arabidopsis thaliana circadian clock. For this, natural variation present in two Recombinant Inbred Lines (RILS) was exploited. The RILS were transformed with a circadian controlled and temperature regulated promoter::reporter construct. Period analysis of this CCR2 reporter after both entrainments revealed a number of Quantitative Trait Loci (QTL) for each collection assayed. The findings suggested that the circadian clockwork after light-dark and temperature entrainment is controlled by both the same, as well as by different, QTLs. Additionally, it was shown that significant allelic interactions modify the period of CCR2. A QTL that was detected, specifically, after temperature entrainment was delineated by fine-mapping procedure. Previous natural-variation studies in the vast majority of pre-existing RILs exploited the variation of two diverse ecotypes, of otherwise rarely used genetic backgrounds. This caveat restricts QTL fine mapping. To confront this disadvantage, six new RILs were generated by pairwise crosses between the four most commonly lab accessions: Columbia, C24, Landsberg erecta, and Wassilewskija. The latter two accessions were previously transformed with CCR2::LUC and used as pollen donor on the other three as a female recipient. All RIL populations were selected for short or long period of CCR2 after light-dark entrainment. Assessment of flowering-time variation under inductive long days, and circadian rhythmicity of CCR2::LUC was measured after light-dark and temperature entrainment. QTL mapping led to the identification of QTLs controlling these two processes. The traits shared some correlations. The majority of the above described QTLs under these two entrainments co-localized with already known components of the circadian oscillator. An alternative approach to physiologically map the role of already known clock genes after temperature-entrainment was thus taken. The transcriptional kinetics of CAB2, CCA1, LHY, TOC1, CCR2, GI, ELF3, and ELF4 were assayed under various light-dark and temperature entrainment protocols. Each promoter displayed unique responses to the different protocols assessed. This suggested that the circadian oscillator is a dynamic mechanism that is able to respond at a variety of signal changes within the ambient environment. The key role of two evening expressed genes, TOC1 and GI, was further defined through the use of genetics, in response to temperature entrainment. A model for GI as a light resettor and TOC1 as a thermal resettor was proposed

Environmental memory from a circadian oscillator:the Arabidopsis thaliana clock differentially integrates perception of photic vs. thermal entrainment

The constraint of a rotating earth has led to the evolution of a circadian clock that drives anticipation of future environmental changes. During this daily rotation, the circadian clock of Arabidopsis thaliana (Arabidopsis) intersects with the diurnal environment to orchestrate virtually all transcriptional processes of the plant cell, presumably by detecting, interpreting, and anticipating the environmental alternations of light and temperature. To comparatively assess differential inputs toward phenotypic and physiological responses on a circadian parameter, we surveyed clock periodicity in a recombinant inbred population modified to allow for robust periodicity measurements after entrainment to respective photic vs. thermal cues, termed zeitgebers. Lines previously thermally entrained generally displayed reduced period length compared to those previously photically entrained. This differential zeitgeber response was also detected in a set of diverse Arabidopsis accessions. Thus, the zeitgebers of the preceding environment direct future behavior of the circadian oscillator. Allelic variation at quantitative trait loci generated significant differences in zeitgeber responses in the segregating population. These were important for periodicity variation dependent on the nature of the subsequent entrainment source. Collectively, our results provide a genetic paradigm for the basis of environmental memory of a preceding environment, which leads to the integrated coordination of circadian periodicity