GIGANTEA and SPINDLY genes linked to the clock pathway that controls circadian characteristics of transpiration in Arabidopsis

Several "clock" genes that regulate the circadian system in Arabidopsis thaliana have been identified. The GIGANTEA (GI) gene has been shown to participate in the circadian system that is linked to overt rhythms in gene expression, leaf movements, hypocotyl elongation, and photoperiodic control of flowering in Arabidopsis. During continuous light (LL), circadian expression patterns in gi-2 mutants show reduced amplitudes and altered period lengths when compared with controls. Rhythms in stomatal function, such as transpiration, have been shown to be endogenous and persist in constant lighting conditions. In order to test for a physiologic variable that might be affected by the circadian clock via the GI gene, we compared circadian characteristics of transpiration between three Arabidopsis mutants (gi-2, spy-4, spy-4/gi-2) and wild-type (WT) controls in synchronized (LD for 2.5d) and free-running (LL for 3d) conditions. Each genotype showed a significant circadian rhythm in LD at p < 0.001, with acrophases located near the middle of the daily 14h L-span, with average amplitudes for WT: 18.9%, gi-2: 16.1%, spy-4: 7.7%, and spy-4/gi-2: 5.3%. On the first day in LL, the circadian amplitude was dramatically reduced to 3.1% for gi-2 compared with WT (11.9%), while amplitudes for spy-4 (6.9%) and spy-4/gi-2 (5.7%) were not significantly changed from LD. The amplitude for gi-2 remained low during days 2 (4.2%) and 3 (2.1%) in LL, while it slowly dampened for the WT (8.6 and 6.6%). The amplitudes for spy-4 (6.6%) and spy-4/gi-2 (5.6%) on day 2 in LL were indistinguishable from the LD span, but finally dampened on day 3 in LL (1.9 and 2.3%, respectively). These data suggest that transpiration is a physiologic variable controlled by a circadian system that involves both the GI and SPY proteins.     

The circadian clock modulates water dynamics and aquaporin expression in Arabidopsis roots

We have developed a plant growth system to analyze water dynamics in the roots of a small model plant, Arabidopsis thaliana, by nuclear magnetic resonance (NMR) microscopic imaging. Using the two-dimensional slice technique, we obtained a series of images with high signal-to-noise ratio indicating the water distribution in the root. To demonstrate light regulation of water transport in the root and involvement of aquaporin gene expression, we visualized the distribution of water in Arabidopsis roots under various light conditions and compared the data with the expression profiles of two aquaporin genes. (1)H-NMR imaging revealed that water content in Arabidopsis roots is lower in the light than in the dark. This diurnal variation in water content was clearly observed in the basal zone of the root. In addition, an autonomous rhythm of water dynamics was observed under continuous light (LL) and darkness (DD). However, the circadian oscillation in water dynamics was obscured in the early-flowering 3 (elf3) mutant under LL. The expression of both the aquaporin genes, AtPIP1;2 and AtPIP2;1, oscillated with the circadian rhythm under LL conditions in wild-type seedlings, but not in the elf3 mutant. These results demonstrate the advantages of our technique for monitoring water dynamics in roots of living Arabidopsis seedlings, and suggest that the circadian clock modulates water dynamics and aquaporin expression.     

Photoperiod sensitivity of the Arabidopsis circadian clock is tissue-specific

Tissue-specific functions of the circadian clock in Arabidopsis have recently been revealed. The vasculature clock shows distinctive gene expression profiles compared to the clock in other tissues under light-dark cycles. However, it has not yet been established whether the vasculature clock also shows unique gene expression patterns that correlate with temperature cycles, another important environmental cue. Here, we detected diel phase of TIMING OF CAB EXPRESSION 1 (TOC1) expression in the vasculature and whole leaf under long-day light-dark cycles and temperature cycles. We found that the vasculature clock had advanced TOC1 phase under light-dark cycles but not under temperature cycles, suggesting that the vasculature clock has lower sensitivity against temperature signals. Furthermore, the phase advancement of TOC1 was seen only under long-day condition but not under short-day condition. These results support our previous conclusion that the circadian clock in vasculature preferentially senses photoperiodic signals.     

A chromatin-dependent mechanism regulates gene expression at the core of the Arabidopsis circadian clock

The mechanisms of circadian clock function in Arabidopsis rely on the complex relationships among core clock components. The current model of the Arabidopsis oscillator comprises a myriad of repressors but the mechanisms responsible for activation remain largely unknown. In our recent studies, we have demonstrated that the rhythms in H3 acetylation (H3ac) and H3K4 trimethylation (H3K4me3) are a key mechanism at the positive arm of the oscillator. H3K4me3 rhythmic accumulation is delayed compared to that of H3ac, which opens the possibility for separate roles for each mark. Indeed, the use of inhibitors that block H3K4me3 accumulation was concomitant with increased clock repressor binding, suggesting that H3K4me3 might control the timing from activation to repression. Plants mis-expressing the histone methyltransferase SET DOMAIN GROUP 2 (SDG2/ATXR3) displayed altered H3K4me3 accumulation, oscillator gene expression and clock repressor binding, suggesting that SDG2/ATXR3 is a key component contributing to proper circadian expression.     

The circadian clock in the retina controls rod-cone coupling

Although rod and cone photoreceptor cells in the vertebrate retina are anatomically connected or coupled by gap junctions, a type of electrical synapse, rod-cone electrical coupling is thought to be weak. Using tracer labeling and electrical recording in the goldfish retina and tracer labeling in the mouse retina, we show that the retinal circadian clock, and not the retinal response to the visual environment, controls the extent and strength of rod-cone coupling by activating dopamine D(2)-like receptors in the day, so that rod-cone coupling is weak during the day but remarkably robust at night. The results demonstrate that circadian control of rod-cone electrical coupling serves as a synaptic switch that allows cones to receive very dim light signals from rods at night, but not in the day. The increase in the strength and extent of rod-cone coupling at night may facilitate the detection of large dim objects.     

Network balance via CRY signalling controls the Arabidopsis circadian clock over ambient temperatures

Circadian clocks exhibit ‘temperature compensation’, meaning that they show only small changes in period over a broad temperature range. Several clock genes have been implicated in the temperature‐dependent control of period in Arabidopsis. We show that blue light is essential for this, suggesting that the effects of light and temperature interact or converge upon common targets in the circadian clock. Our data demonstrate that two cryptochrome photoreceptors differentially control circadian period and sustain rhythmicity across the physiological temperature range. In order to test the hypothesis that the targets of light regulation are sufficient to mediate temperature compensation, we constructed a temperature‐compensated clock model by adding passive temperature effects into only the light‐sensitive processes in the model. Remarkably, this model was not only capable of full temperature compensation and consistent with mRNA profiles across a temperature range, but also predicted the temperature‐dependent change in the level of LATE ELONGATED HYPOCOTYL, a key clock protein. Our analysis provides a systems‐level understanding of period control in the plant circadian oscillator.     

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.     

Beyond Arabidopsis: The circadian clock in non-model plant species

Circadian clocks allow plants to temporally coordinate many aspects of their biology with the diurnal cycle derived from the rotation of Earth on its axis. Although there is a rich history of the study of clocks in many plant species, in recent years much progress in elucidating the architecture and function of the plant clock has emerged from studies of the model plant, Arabidopsis thaliana. There is considerable interest in extending this knowledge of the circadian clock into diverse plant species in order to address its role in topics as varied as agricultural productivity and the responses of individual species and plant communities to global climate change and environmental degradation. The analysis of circadian clocks in the green lineage provides insight into evolutionary processes in plants and throughout the eukaryotes.     

Hypothermia modulates circadian clock gene expression in lizard peripheral tissues

The molecular mechanisms whereby the circadian clock responds to temperature changes are poorly understood. The ruin lizard Podarcis sicula has historically proven to be a valuable vertebrate model for exploring the influence of temperature on circadian physiology. It is an ectotherm that naturally experiences an impressive range of temperatures during the course of the year. However, no tools have been available to dissect the molecular basis of the clock in this organism. Here, we report the cloning of three lizard clock gene homologs (Period2, Cryptochrome1, and Clock) that have a close phylogenetic relationship with avian clock genes. These genes are expressed in many tissues and show a rhythmic expression profile at 29 degrees C in light-dark and constant darkness lighting conditions, with phases comparable to their mammalian and avian counterparts. Interestingly, we show that at low temperatures (6 degrees C), cycling clock gene expression is attenuated in peripheral clocks with a characteristic increase in basal expression levels. We speculate that this represents a conserved vertebrate clock gene response to low temperatures. Furthermore, these results bring new insight into the issue of whether circadian clock function is compatible with hypothermia.     

Acute light exposure suppresses circadian rhythms in clock gene expression

Light can induce arrhythmia in circadian systems by several weeks of constant light or by a brief light stimulus given at the transition point of the phase response curve. In the present study, a novel light treatment consisting of phase advance and phase delay photic stimuli given on 2 successive nights was used to induce circadian arrhythmia in the Siberian hamster ( Phodopus sungorus). We therefore investigated whether loss of rhythms in behavior was due to arrhythmia within the suprachiasmatic nucleus (SCN). SCN tissue samples were obtained at 6 time points across 24 h in constant darkness from entrained and arrhythmic hamsters, and per1, per2 , bmal1, and cry1 mRNA were measured by quantitative RT-PCR. The light treatment eliminated circadian expression of clock genes within the SCN, and the overall expression of these genes was reduced by 18% to 40% of entrained values. Arrhythmia in per1, per2, and bmal1 was due to reductions in the amplitudes of their oscillations. We suggest that these data are compatible with an amplitude suppression model in which light induces singularity in the molecular circadian pacemaker.