Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Age-dependent changes of the ion content and the circadian leaf movement period in thePhaseolus pulvinus

The circadian movement of the lamina of primary leaves ofPhaseolus coccineus L. depends on circadian changes of the K⁺, Cl^- and (depending on the Cl^- availability) malate content in the swelling and shrinking motor cells of the laminar pulvinus. After sowing in soil, the laminar pulvinus develops within about 26 days. When the leaves emerge from the soil (about 6 days after sowing) and the pulvinus starts with the diurnal movement (about 9 days after sowing) the pulvinar dimensions are about half of those of the mature pulvinus. The anatomical structure, however, is basically the same as in the developed pulvinus. In soil-grown plants, the K⁺, Cl^- and malate content as well as the period length of the circadian leaf movement rhythm change in the developing pulvinus. In the embryo of the dry seed, the Cl^- content is low (0.03 mmol g^-1 DW), the K⁺ content, however, 22-fold higher than the Cl^- content. When the leaves emerge from the soil, the pulvinar K⁺ and Cl^- content is the same as in the whole embryo of the dry seed. In the developing pulvinus the K⁺ content increases by a factor of 2 and the Cl^- content by a factor of 41 in the mature pulvinus. The pulvinar malate content increases between the 6th and 10th day from about 40 to 180Μmol g^-1 DW, then decreases until the 17th day and remains thereafter on a low level (around 80 Μmol g^-1 DW). These results indicate that the Cl^- availability increases in the developing pulvinus with age. It explains furthermore why in young leaves malate was found as counterion to K⁺ in the osmotic leaf movement motor, in older ones, however, Cl^-. The circadian leaf movement starts 9 days after sowing. The period length decreases during the development of the pulvinus from 31.3 to 28.6 h in leaves of intact soil-grown plants. In leaves which were cut from the plants and immersed with their petioles in distilled water, the age dependent decrease of the period length is also found. However, the period lengths are shorter by more than 1 h than in the leaves of intact plants. The increasing Cl^- availability in the developing pulvinus does not seem to be the cause for the age dependent shortening of the period length, because the period length in 22 days old Cl^- deprived pulvini is the same as in 22 days old pulvini with a high Cl^- content.     

On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

Cell Division Cycles and Orcadian Oscillators in Euglena

The algal flagellate Euglena grown photoautotrophically in L:D 3:3 displays a circadian rhythm of cell division. Oscillatory models for cell cycle (CDC) control (particularly those of the limit cycle variety) include the property of phase perturbation, or resetting. This prediction has been tested in synchronous cultures in which the free-running rhythm has been scanned by 3-hr light signals. A strong (Type 0) phase response curve (PRC), yielding both advances and delays as great as 15 hr, has been derived. A second prediction of the limit cycle model is that there exists a pulse of a critical intensity, which, if given at one specific phase of the rhythm (the singularity point), should result in a phaseless, motionless state in which the rhythmicity disappears. Such a point has been found in Euglena in the late subjective night for light pulses having an intensity ranging from 40 to 700 Ix. Finally, circadian oscillators typically display temperature-compensated period lengths within the physiological range of steady-state temperatures, although the length of the CDC is commonly thought to be highly temperature dependent. We have found that over a range of at least 10°C, the period of the division rhythm is only slightly affected, exhibiting a Q₁₀ of about 1.05-1.20. These observations, therefore, collectively implicate a circadian oscillator in the control of the CDC.     

Many Circadian Oscillators Regulate Developmental and Behavioural Events in the Flesh-Fly, Sarcophaga Argyrostoma

In the flesh-fly, Sarcophaga argyrostoma, the initiation of larval wandering, pupal eclosion, and the induction of pupal diapause by seasonal changes in night length, are all regulated by circadian oscillators. They differ, however, in several respects. The rhythm of larval wandering shows a free-running period (S) of about 20 hr and a steady-state phase-relationship to the light cycle (±) in which maximum activity occurs at dusk or in the night; that for pupal eclosion shows ± close to 24 hr and ± close to dawn; and that for diapause induction ± longer than 24 hr and a photoinducible phase (φ_i) late in the subjective night. The three oscillators are, therefore, considered to be functionally separate. In addition, adult locomotor activity, the deposition of cuticular growth layers on thoracic apodemes, and the duration of larval wandering, are possibly regulated by further, distinct, oscillators. The circadian system in S. argyrostoma, therefore, contains at least three, and probably as many as six, known circadian pacemakers.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

蓝光和蛋白酶体抑制剂MG132处理对蛹虫草Cmfrq、Cmwc-1和Cmwc-2转录水平的影响

生物钟的节律振荡器主要成分之间的关系构成了转录-翻译负反馈环,并以此调控生物体的生理生化反应和生长发育等.以不同时间的蓝光照射和蛋白酶体抑制剂MG132处理蛹虫草菌丝体,通过实时荧光PCR分析其中节律振荡器主要成分的3个基因Cmfrq、Cmwc-1和Cmwc-2转录水平变化,以期确定3个基因在蛹虫草中的相互关系和变化规...     

Circadian Activity Rhythm of the House Fly Continues after Optic Tract Severance and Lobectomy

Under constant conditions, locomotor activity in about 50% of 63 adult Musca domestica continued to be rhythmic after bilateral severance of optic tracts or bilateral lobectomy. Apparently, the optic lobes of Musca do not contain the oscillator for rhythmic control of locomotor activity as has been proposed for other insects. In 20% of the individuals, several circadian components of activity rhythms were found after operation indicating a role of the optic lobes in the coupling of oscillators. The remaining 30% of the flies with severed optic tracts appeared to be arrhythmic. Most of these flies had vacuolized tissue in the central brain. However, disruption of rhythmicity did not correlate with a common pattern of degeneration. Therefore no conclusions can be drawn as to the localization of the circadian control of locomotor activity in the brain. Flies showing an arrhythmic activity pattern could still be synchronized by LD cycles. Activity did not occur solely during the light period as is the case in controls; but was phase delayed by about 6 hr towards the dark period. Since all flies with severed optic tracts could be synchronized by LD cycles, Musca domestica must possess extraocular photoreceptors.     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Light- and Clock-Controlled Leaflet Movements in Samanea saman*: A Physiological,Biophysical and Biochemical Analysis**

Leaflet movements in the legume Samanea saman are under joint control by light and a circadian oscillator. The movements are driven by massive fluxes of K⁺, Cl^?, and H⁺ through pulvinar motor cell membranes. Light and the oscillator affect leaflet movements by altering the activity of ion transport systems. Some effects of light on ion transport may be mediated by the phosphatidylinositol (PI) cycle, since brief irradiation of the pulvinus with white light accelerates PI turnover.     

An Experimental Analysis and Comparison of Three Rhythms of Movements in Bean (Phaseolus Vulgaris L.)

Three types of rhythmic movements of Phaseolus vulgaris L. (pole beans) were examined collectively and their characteristics compared. Although the ultradian rhythms of shoot circumnutation and leaf movement, as well as the circadian rhythm of leaf movement, occurred simultaneously, each rhythm could be expressed independently of the other two. Shoot circumnutation and ultradian leaf movements displayed the same period (80 min at 25°C and Q₁₀⋍2), while the period of the circadian leaf movements was not temperature dependent (Q₁₀⋍1). Interaction into the plant between two ultradian rhythms (shoot circumnutation and ultradian leaf movement) with the same period and coexistence in the pulvinus of an ultradian with a circadian rhythm are discussed.     

Probing the circadian oscillator of a mammal by two-pulse perturbations

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129-136, 2000)     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Correlation of the Period Length of Circadian Rhythms with the Length of Immaturity in Paramecium bursaria

The circadian photoaccumulation rhythm of thirty strains of Paramecium bursaria collected at different places in Japan and China were measured with a microcomputer assisted data collection apparatus. Although most strains showed a period of 23-26 hours in LL, we found two strains of conspicuously different periods; a short period strain (UK1, 21.8 hr) and a long period strain (T316, 28.7 hr). F1 progeny from a cross between the short and the long period strains showed an intermediate period of about 24.7 hours (range 22.5-25.8 hr). The character was not distributed in a Mendelian ratio among the F1 progeny. We isolated a mutant (E2) with short period (21.8 hr) from the stock strain Kz1 by treatment with nitrosoguanidine (MNNG). The progeny of crosses between E2 and UK1, and between E2 and T316 exhibited the short period and the normal period phenotype respectively. Moreover, the progeny from a cross between E2 and a wild type strain (Sj2w) became sexually mature about 25 fissions after conjugation. This length of immaturity is much shorter than that of the progeny from wild type strains (about 50 fissions). This early maturation character was inherited to progeny in a Mendelian ratio. Homozygotes for the early maturation allele (EM2) exhibited mating ability about 15 fissions after conjugation. These data suggest that there is a correlation between the period length of the circadian rhythm and the length of immaturity after conjugation in Paramecium bursaria.     

Expression of clock genes in human peripheral blood mononuclear cells throughout the sleep/wake and circadian cycles

The rhythmic expression of circadian clock genes in the neurons of the suprachiasmatic nucleus (SCN) underlies the manifestation of endogenous circadian rhythmicity in behavior and physiology. Recent evidence demonstrating rhythmic clock gene expression in non-SCN tissues suggests that functional clocks exist outside the central circadian pacemaker of the brain. In this investigation, the nature of an oscillator in peripheral blood mononuclear cells (PBMCs) is evaluated by assessing clock gene expression throughout both a typical sleep/wake cycle (LD) and during a constant routine (CR). Six healthy men and women aged (mean±SEM) 23.7±1.6 yrs participated in this five-day investigation in temporal isolation. Core body temperature and plasma melatonin concentration were measured as markers of the central circadian pacemaker. The expression of HPER1, HPER2, and HBMAL1 was quantified in PBMCs sampled throughout an uninterrupted 72 h period. The core body temperature minimum and the midpoint of melatonin concentration measured during the CR occurred 2:17±0:20 and 3:24 ±0:09 h before habitual awakening, respectively, and were well aligned to the sleep/wake cycle. HPER1 and HPER2 expression in PBMCs demonstrated significant circadian rhythmicity that peaked early after wake-time and was comparable under LD and CR conditions. HBMAL1 expression was more variable, and peaked in the middle of the wake period under LD conditions and during the habitual sleep period under CR conditions. For the first time, bi-hourly sampling over three consecutive days is used to compare clock gene expression in a human peripheral oscillator under different sleep/wake conditions.     

Further studies on the involvement of the circadian system in photoperiodic control of antifreeze protein production in the beetle Dendroides canadensis

The role of circadian rhythmicity in the photoperiodic time measuring processes regulating antifreeze protein production in the beetle Dendroides canadensis was further investigated. Using “T” experiments larvae were exposed to environmental light cycle periods close to the period length of the endogenous circadian oscillator. The following light cycles were employed: light/dark 8/13, 8/14, 8/16, 8/18 and 8/19 corresponding to period lengths of 21, 22, 24, 26 and 27 h. Larvae maintained in cycles equal to or less than 24 h displayed a characteristic short-day response, showing significantly (P < 0.01) greater antifreeze protein activity than did those measured on the day of collection in late summer. In contrast, a long-day response was observed in larvae maintained under a 26- or 27-h light cycle in that antifreeze protein activity did not differ from that measured on the initial collection date.

The role of photoperiod and temperature in influencing the photoperiodic timing processes were examined with a series of resonance experiments. The first group consisted of a 24, 36, 48, 60 or 72-h light cycle, each with an 8-h photophase at temperatures of 20 or 17°C. Rhythmic increases in antifreeze protein levels at intervals of 24 h occurred under both temperatures. However, the lower temperature displaced the resonance curve in the vertical direction (i.e. increasing % population response) and reduced the difference between peaks and troughs on the resonance curve. Resonance experiments incorporating a 14-h photophase resulted in low antifreeze protein activity under all conditions except a 36-h light cycle in which a 67% induction was observed.

Eight hour resonance experiments were also conducted with D. canadensis collected in early spring to determine whether the circadian system participates in the photoperiodic timing processes influencing the spring termination of antifreeze protein production. Positive resonance results were obtained in that only larvae maintained in cycles of 36 and 60 h displayed significantly (P < 0.01) lower antifreeze activity when compared to animals on the initial collection date.

The combined results emphasize the involvement of the circadian system in the photoperiodic control of antifreeze protein production by D. canadensis during the fall and spring. Furthermore, the induction of antifreeze protein production is a function of light cycle and its waveform (photoperiod). Temperature appears to modify the photoperiodic response in some manner involving the photoperiodic time measuring processes. It is concluded that the photoperiodic response of antifreeze protein production by D. canadensis is dependent upon the entrainment of the circadian system by the light cycle.     

Mechanics and resonance of the cyanobacterial circadian oscillator

Recent experiments elucidated the structure and function of the cyanobacterial circadian oscillator, which is driven by sunlight intensity variation and therefore by Earth's rotation. It is known that cyanobacteria appeared about 3.5 billion years ago and that Earth's rotational speed is continuously decreasing because of tidal friction. What is the effect of the continuous slowdown of Earth's rotation on the operation of the cyanobacterial oscillator? To answer this question we derived the oscillator's equation of motion directly from experimental data, coupled it with Earth's rotation and computed its natural periods and its resonance curve. The results show that there are two resonance peaks of the “cyanobacterial oscillator-rotating Earth” system, indicating that cyanobacteria used more efficiently the solar energy during the geological period in which the day length varied from about 11 to 15 h and make more efficient use of solar energy at the geological period which started with a day length of 21 h and will end at a day length of 28 h.     

The Circadian Rhythm of Leaf Movement of Coleus blumei x C. frederici, a Short Day Plant. II. The Effects of Light and Temperature Signals

The phase response curve for the circadian rhythm of leaf movement of Coleus blumei x C. frederici, a short day plant, is generally similar to those reported for other organisms. An increase in the duration of the light signal caused an increase in the extreme values of the phase response curve and shortened the time for transition from maximum delays to maximum advances. Experiments with 2 light signals showed that the overt rhythm of leaf movement represents the rhythm of the light sensitive oscillator even during the transient period that followed the first light signal. A temperature decrease of 7° for 8 hr caused only a transient phase shift in the following 2 cycles but not in the steady state. The combination of such a temperature decrease and a light signal showed that only the overt rhythm of leaf movement was disturbed by the temperature decrease whereas the light sensitive oscillator was free running. A temperature decrease of 11° for 10 hr caused a steady state phase shift and affected the light sensitive oscillator as well.