Focusing on the nuclear and subnuclear dynamics of light and circadian signalling

Circadian clocks provide organisms the ability to synchronize their internal physiological responses with the external environment. This process, termed entrainment, occurs through the perception of internal and external stimuli. As with other organisms, in plants, the perception of light is a critical for the entrainment and sustainment of circadian rhythms. Red, blue, far‐red, and UV‐B light are perceived by the oscillator through the activity of photoreceptors. Four classes of photoreceptors signal to the oscillator: phytochromes, cryptochromes, UVR8, and LOV‐KELCH domain proteins. In most cases, these photoreceptors localize to the nucleus in response to light and can associate to subnuclear structures to initiate downstream signalling. In this review, we will highlight the recent advances made in understanding the mechanisms facilitating the nuclear and subnuclear localization of photoreceptors and the role these subnuclear bodies have in photoreceptor signalling, including to the oscillator. We will also highlight recent progress that has been made in understanding the regulation of the nuclear and subnuclear localization of components of the plant circadian clock.     

Diurnal regulation of plant growth

Life occurs in an ever-changing environment. Some of the most striking and predictable changes are the daily rhythms of light and temperature. To cope with these rhythmic changes, plants use an endogenous circadian clock to adjust their growth and physiology to anticipate daily environmental changes. Most studies of circadian functions in plants have been performed under continuous conditions. However, in the natural environment, diurnal outputs result from complex interactions of endogenous circadian rhythms and external cues. Accumulated studies using the hypocotyl as a model for plant growth have shown that both light signalling and circadian clock mutants have growth defects, suggesting strong interactions between hypocotyl elongation, light signalling and the circadian clock. Here, we review evidence suggesting that light, plant hormones and the circadian clock all interact to control diurnal patterns of plant growth.     

Diurnal and annual rhythms in trees

Trees, perennial phanerophytes, display a rich variety of rhythmic phenomena. These are either due to exclusive environmental entrainment or due to the functioning of endogenous oscillators independent of the environment. Both types of rhythms are covered in this review. Purely environment controlled rhythms may be considered as a prelude to endogenous rhythms. Environment controlled rhythms discussed are (i) the diurnal rhythms of nyctinastic and heliotropic leaf movements and oscillatory phenomena of photosynthesis, such as the midday depression and Crassulacean acid metabolism (CAM), and (ii) the annual rhythms of annual growth ring formation, autumnal leaf senescence, over wintering mechanisms and flowering. Among the diurnal rhythms, nyctinastic movements and CAM are also free-running endogenous rhythms showing the operation of circadian clocks in trees. In leaf senescence, over wintering, and flowering control, photoperiod sensing is involved which suggests the participation of endogenous clocks. A question asked is if diurnal and annual rhythms are mechanistically correlated. Evidently, phenological phenomena based on photoperiodism (as dependent on measurement of night length) are co-ordinately regulated by the phytochrome system and the circadian clocks and many aspects of annual developments and over wintering are linked to photoperiodism. The existence in trees of circadian clock genes as known to be anchored in the genome of A. thaliana can be assessed by attempts of alignment with the sequenced genome of Populus or by isolating cDNA clones from trees to check them against the genome of A. thaliana. At extreme latitudes near the equator and north of the polar circle trees also display photoperiod-independent phenological phenomena. In the polar region, total irradiance of red and far red light could possibly be involved and the signalling pathway then involves phytochrome, and thus, may still be similar to that of photoperiodism. At the equator, total daily light irradiance received or sensing the dynamics of daily changes in solar irradiance are essential and it remains enigmatic whether signalling cascades are either attached to the circadian clocks in a still unknown way or totally independent of circadian clocks.     

Entrainment of the <Emphasis Type="Italic">Arabidopsis</Emphasis> Circadian Clock

The rising and setting of the sun marks a transition between starkly contrasting environmental conditions for vegetative life. Given these differing diurnal and nocturnal environmental factors and the inherent regularity of the transition between the two, it is perhaps unsurprising that plants have developed an internal timing mechanism (known as a circadian clock) to allow modulation of gene expression and metabolism in response to external cues. Entrainment of the circadian clock, primarily via the detection of changes in light and temperature, maintains synchronization between the surrounding environment and the endogenous clock mechanism. In this review, recent advances in our understanding of the molecular workings of the plant circadian clock are discussed as are the input pathways necessary for entrainment of the clock machinery.     

Circadian effects of light no brighter than moonlight

In mammals, light entrains endogenous circadian pacemakers by inducing daily phase shifts via a photoreceptor mechanism recently discovered in retinal ganglion cells. Light that is comparable in intensity to moonlight is generally ineffective at inducing phase shifts or suppressing melatonin secretion, which has prompted the view that circadian photic sensitivity has been titrated so that the central pacemaker is unaffected by natural nighttime illumination. However, the authors have shown in several different entrainment paradigms that completely dark nights are not functionally equivalent to dimly lit nights, even when nighttime illumination is below putative thresholds for the circadian visual system. The present studies extend these findings. Dim illumination is shown here to be neither a strong zeitgeber, consistent with published fluence response curves, nor a potentiator of other zeitgebers. Nevertheless, dim light markedly alters the behavior of the free-running circadian pacemaker. Syrian hamsters were released from entrained conditions into constant darkness or dim narrowband green illumination (~0.01 lx, 1.3 x 10(-9) W/cm(2), peak lambda = 560 nm). Relative to complete darkness, constant dim light lengthened the period by ~0.3 h and altered the waveform of circadian rhythmicity. Among animals transferred from long day lengths (14 L:10 D) into constant conditions, dim illumination increased the duration of the active phase (alpha) by ~3 h relative to complete darkness. Short day entrainment (8 L:16 D) produced initially long alpha that increased further under constant dim light but decreased under complete darkness. In contrast, dim light pulses 2 h or longer produced effects on circadian phase and melatonin secretion that were small in magnitude. Furthermore, the amplitude of phase resetting to bright light and nonphotic stimuli was similar against dimly lit and dark backgrounds, indicating that the former does not directly amplify circadian inputs. Dim illumination markedly alters circadian waveform through effects on alpha, suggesting that dim light influences the coupling between oscillators theorized to program the beginning and end of subjective night. Physiological mechanisms responsible for conveying dim light stimuli to the pacemaker and implications for chronotherapeutics warrant further study.     

Does pupil constriction under blue and green monochromatic light exposure change with age?

Many nonvisual functions are regulated by light through a photoreceptive system involving melanopsin-expressing retinal ganglion cells that are maximally sensitive to blue light. Several studies have suggested that the ability of light to modulate circadian entrainment and to induce acute effects on melatonin secretion, subjective alertness, and gene expression decreases during aging, particularly for blue light. This could contribute to the documented changes in sleep and circadian regulatory processes with aging. However, age-related modification in the impact of light on steady-state pupil constriction, which regulates the amount of light reaching the retina, is not demonstrated. We measured pupil size in 16 young (22.8±4 years) and 14 older (61±4.4 years) healthy subjects during 45-second exposures to blue (480 nm) and green (550 nm) monochromatic lights at low (7×10(12) photons/cm2/s), medium (3×10(13) photons/cm2/s), and high (10(14) photons/cm2/s) irradiance levels. Results showed that young subjects had consistently larger pupils than older subjects for dark adaptation and during all light exposures. Steady-state pupil constriction was greater under blue than green light exposure in both age groups and increased with increasing irradiance. Surprisingly, when expressed in relation to baseline pupil size, no significant age-related differences were observed in pupil constriction. The observed reduction in pupil size in older individuals, both in darkness and during light exposure, may reduce retinal illumination and consequently affect nonvisual responses to light. The absence of a significant difference between age groups for relative steady-state pupil constriction suggests that other factors such as tonic, sympathetic control of pupil dilation, rather than light sensitivity per se, account for the observed age difference in pupil size regulation. Compared to other nonvisual functions, the light sensitivity of steady-state pupil constriction appears to remain relatively intact and is not profoundly altered by age.     

Cryptochromes: enabling plants and animals to determine circadian time

Cryptochromes are flavin-containing blue light photoreceptors related to photolyases-they are found in both plants and animals and have recently been described for bacteria. In plants, cryptochromes perform a variety of functions including the entrainment of circadian rhythms. They serve a similar role in Drosophila and mammals, where the cryptochromes also perform an additional function as an essential component of the circadian clock.     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781-799, 2001)     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781–799, 2001)     

Retinal and extraretinal photoreceptors mediate entrainment of the circadian locomotor rhythm in lizards

Summary The circadian locomotor activity rhythms of 7 species of lizards can readily be entrained (synchronized) toLD12: 12 (30–50 lux: 0) fluorescent light cycles after complete surgical removal of both eyes. Removal of the parietal eye and pineal organ does not prevent entrainment of blinded lizards. Appropriate control experiments established that lightper se, and not low amplitude temperature cycles or other obvious environmental variables, was the entraining stimulus for blinded lizards. In some cases, blocking the penetration of light to the brains of blinded lizards caused them to free-run (express their endogenous circadian rhythm) in the presence of a dim green light cycle, to which they had previously entrained, suggesting that the brain is the site of the extraretinal photoreceptor(s) mediating entrainment. The extraretinal photoreceptor(s) is capable of intensity discrimination since changing the intensity of aLD 12: 12 fluorescent light cycle caused a change in the phase-relationship between the entrained activity rhythm and the light cycle in a blinded gekko. The lateral eyes are also involved in mediating entrainment since removal of the lateral eyes of thoseSceloporus olivaceus which previously entrained to a dim green light cycle [LD 12: 12 (0.05 lux: 0)] caused them to free-run. Also, blinding had noticeable effects on the entrained activity patterns of some species of lizards.I thank Michael Menaker, Jeffrey Elliott, Sue Binkley, Joseph Silver, Ed Kluth, George Wyche, Bruee Rouse, Nancy Leshikar, Lili Mostafavi, Janet Alvis, Celeste Cromack, A. L. Mackey and Jean Rogers for their suggestions and technical assistance. Support for this work was provided by NIH grant HD-03803-02 (to M. Menaker); NSP grant GB-8138 (to M. Menaker); NSF traineeship GZ-1336 (to H. Underwood); and MH traineeship 5T01GM00836-09 (to H. Underwood).     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

Mice Lacking the p75NGFR Receptor Exhibit Abnormal Responses to Light

A role for nerve growth factor (NGF) in the mammalian circadian system was assessed in mice using a line of gene targeted animals carrying a null mutation at the low affinity p75 NGFR receptor locus. Receptor-deficient mice exhibited positive phase angles of entrainment to 24 h light-dark cycles. In constant dark, circadian rhythms of behavior were normal but phase shifts induced by brief pulses of light were significantly decreased. These results support the hypothesis that NGF, through an action mediated at least in part by p75 NGFR receptors in the SCN, is involved in the regulation of circadian responses to light.     

Mice Lacking the p75NGFR Receptor Exhibit Abnormal Responses to Light

A role for nerve growth factor (NGF) in the mammalian circadian system was assessed in mice using a line of gene targeted animals carrying a null mutation at the low affinity p75 NGFR receptor locus. Receptor-deficient mice exhibited positive phase angles of entrainment to 24 h light-dark cycles. In constant dark, circadian rhythms of behavior were normal but phase shifts induced by brief pulses of light were significantly decreased. These results support the hypothesis that NGF, through an action mediated at least in part by p75 NGFR receptors in the SCN, is involved in the regulation of circadian responses to light.     

The inner clock—Blue light sets the human rhythm

Visible light synchronizes the human biological clock in the suprachiasmatic nuclei of the hypothalamus to the solar 24‐hour cycle. Short wavelengths, perceived as blue color, are the strongest synchronizing agent for the circadian system that keeps most biological and psychological rhythms internally synchronized. Circadian rhythm is important for optimum function of organisms and circadian sleep–wake disruptions or chronic misalignment often may lead to psychiatric and neurodegenerative illness. The beneficial effect on circadian synchronization, sleep quality, mood, and cognitive performance depends not only on the light spectral composition but also on the timing of exposure and its intensity. Exposure to blue light during the day is important to suppress melatonin secretion, the hormone that is produced by the pineal gland and plays crucial role in circadian rhythm entrainment. While the exposure to blue is important for keeping organism's wellbeing, alertness, and cognitive performance during the day, chronic exposure to low‐intensity blue light directly before bedtime, may have serious implications on sleep quality, circadian phase and cycle durations. This rises inevitably the need for solutions to improve wellbeing, alertness, and cognitive performance in today's modern society where exposure to blue light emitting devices is ever increasing.      

What makes the Arabidopsis clock tick on time? A review on entrainment

Entrainment, the synchronization of a circadian clock with the external environment, is a crucial step in daily life. Although many signals contribute to entrainment, light and temperature are typically the strongest resetting cues. Much progress has been made concerning light resetting in the model plant Arabidopsis thaliana. Multiple photoreceptors (phytochromes, cryptochromes, LOV-domain proteins) are involved in light perception. The clock genes CCA1, LHY and TOC1 are all probable targets of light signalling, although the details of these pathways are not completely established. Temperature can entrain the clock, but little is known about the mechanism underlying this resetting; no obvious clock gene candidate for temperature resetting has been identified. Although circadian research has emphasized oscillations in free-running conditions, in the real world the circadian clock is entrained. During entrainment, short or long period mutants exhibit a 24-h period, but a mutant phenotype is often manifested as an altered phase relationship with the entraining cycle; short and long period mutants show leading and lagging phases, respectively, and this may be detrimental under some conditions. Arrhythmic CCA1-overexpressing plants display increased lethality under very short photoperiods, consistent with the circadian clock being of adaptive significance to life on a rotating world.