Molecular basis of flowering under natural long-day conditions in Arabidopsis.

Plants sense light and temperature changes to regulate flowering time. Here, we show that expression of the Arabidopsis florigen gene, FLOWERING LOCUS T (FT), peaks in the morning during spring, a different pattern than we observe in the laboratory. Providing our laboratory growth conditions with a red/far-red light ratio similar to open-field conditions and daily temperature oscillation is sufficient to mimic the FT expression and flowering time in natural long days. Under the adjusted growth conditions, key light signalling components, such as phytochrome A and EARLY FLOWERING 3, play important roles in morning FT expression. These conditions stabilize CONSTANS protein, a major FT activator, in the morning, which is probably a critical mechanism for photoperiodic flowering in nature. Refining the parameters of our standard growth conditions to more precisely mimic plant responses in nature can provide a powerful method for improving our understanding of seasonal response.

Light and circadian regulation of clock components aids flexible responses to environmental signals.

The circadian clock measures time across a 24 h period, increasing fitness by phasing biological processes to the most appropriate time of day. The interlocking feedback loop mechanism of the clock is conserved across species; however, the number of loops varies. Mathematical and computational analyses have suggested that loop complexity affects the overall flexibility of the oscillator, including its responses to entrainment signals. We used a discriminating experimental assay, at the transition between different photoperiods, in order to test this proposal in a minimal circadian network (in Ostreococcus tauri) and a more complex network (in Arabidopsis thaliana). Transcriptional and translational reporters in O. tauri primarily tracked dawn or dusk, whereas in A. thaliana, a wider range of responses were observed, consistent with its more flexible clock. Model analysis supported the requirement for this diversity of responses among the components of the more complex network. However, these and earlier data showed that the O. tauri network retains surprising flexibility, despite its simple circuit. We found that models constructed from experimental data can show flexibility either from multiple loops and/or from multiple light inputs. Our results suggest that O. tauri has adopted the latter strategy, possibly as a consequence of genomic reduction.

Spontaneous spatiotemporal waves of gene expression from biological clocks in the leaf.

The circadian clocks that drive daily rhythms in animals are tightly coupled among the cells of some tissues. The coupling profoundly affects cellular rhythmicity and is central to contemporary understanding of circadian physiology and behavior. In contrast, studies of the clock in plant cells have largely ignored intercellular coupling, which is reported to be very weak or absent. We used luciferase reporter gene imaging to monitor circadian rhythms in leaves of Arabidopsis thaliana plants, achieving resolution close to the cellular level. Leaves grown without environmental cycles for up to 3 wk reproducibly showed spatiotemporal waves of gene expression consistent with intercellular coupling, using several reporter genes. Within individual leaves, different regions differed in phase by up to 17 h. A broad range of patterns was observed among leaves, rather than a common spatial distribution of circadian properties. Leaves exposed to light-dark cycles always had fully synchronized rhythms, which could desynchronize rapidly. After 4 d in constant light, some leaves were as desynchronized as leaves grown without any rhythmic input. Applying light-dark cycles to such a leaf resulted in full synchronization within 2-4 d. Thus, the rhythms of all cells were coupled to external light-dark cycles far more strongly than the cellular clocks were coupled to each other. Spontaneous desynchronization under constant conditions was limited, consistent with weak intercellular coupling among heterogeneous clocks. Both the weakness of coupling and the heterogeneity among cells are relevant to interpret molecular studies and to understand the physiological functions of the plant circadian clock.

Data assimilation constrains new connections and components in a complex, eukaryotic circadian clock model.

Circadian clocks generate 24-h rhythms that are entrained by the day/night cycle. Clock circuits include several light inputs and interlocked feedback loops, with complex dynamics. Multiple biological components can contribute to each part of the circuit in higher organisms. Mechanistic models with morning, evening and central feedback loops have provided a heuristic framework for the clock in plants, but were based on transcriptional control. Here, we model observed, post-transcriptional and post-translational regulation and constrain many parameter values based on experimental data. The model's feedback circuit is revised and now includes PSEUDO-RESPONSE REGULATOR 7 (PRR7) and ZEITLUPE. The revised model matches data in varying environments and mutants, and gains robustness to parameter variation. Our results suggest that the activation of important morning-expressed genes follows their release from a night inhibitor (NI). Experiments inspired by the new model support the predicted NI function and show that the PRR5 gene contributes to the NI. The multiple PRR genes of Arabidopsis uncouple events in the late night from light-driven responses in the day, increasing the flexibility of rhythmic regulation.