Light-dark Regime Research Articles

Anthocyanin production in cultured cells of Aralia cordata Thunb.

High anthocyanin-producing cell lines, which were grown in a dark or in a light-dark regime, were selected from callus cultures initiated from stem and leaf tissues of Aralia cordata Thunb. by small-cell-aggregate selection. To verify the optimum culture conditions for anthocyanin production, cells were tested by changing the various basal media, sucrose concentration and nitrogen source and concentration. Good growth was obtained in the dark on Linsmaier-Skoog's basal medium containing 1.0 mg l-1 2,4-d and 0.1 mg l-1 kinetin, 2% (w/v) sucrose and full strength of nitrogen concentration. However, the highest anthocyanin yield (10.3% dry wt) was obtained in the dark on B5 medium containing 1.0 mg l-1 2,4-d and 0.1 mg l-1 kinetin. Our results suggested that it has became feasible to find the most effective conditions for cell growth and anthocyanin production by optimizations of the nitrogen concentration and the ratio of NH4 + to NO3 - in the medium.

Regulation of nitrogenase activity in relation to the light-dark regime in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067

SUMMARY: A periodicity in nitrogen fixation potential with respect to the light-dark regime was studied in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067. During a 12 h light/12 h dark cycle, potential nitrogenase activity measured by acetylene reduction in the light was insignificant in the dark period, but developed after illumination for 1 to 3 h. Maximum nitrogenase activity was found at the middle of the light period, and activity decreased near the end of the light period. Manipulation of the length of the light and dark periods, and use of the glutamine synthetase inhibitor L-methionine sulphoximine, led to the conclusion that (1) the periodicity in activity was not attributable to an endogenous rhythm, (2) development and maintenance of nitrogenase activity in Trichodesmium was regulated by the light period, and (3) the decrease in activity at the end of the light period was due to the accumulation of an intermediate(s) in nitrogen metabolism. The nitrogenase Fe- and MoFe-proteins were always present despite the changes in nitrogenase activity associated with the light-dark cycle. However, a change in apparent molecular mass of the Fe-protein on SDS-PAGE correlated with the change in nitrogenase activity. The results indicate that changes of nitrogenase activity in Trichodesmium under a light-dark regime can be attributed to activation and deactivation of the Fe-protein, and that the activation of the protein depends on light.

Circadian organization of locomotion activity in the haematophagous bug Triatoma infestans

Daily locomotor activity of Triatoma infestans adults exhibits two major components, one at the second hour of the scotophase and a second one at the beginning of the photophase. The occurrence of peaks can be ascribed to food and refuge search at dusk and dawn, respectively. Both maxima appear under discrete light-dark regimes (12 h light-12 h dark, 25:0 lx) at constant temperature, as well as under discrete ligh-low temperature cycles 12 h at 30°C-12 h at 20°C in continuous light or in darkness. Under constant darkness, both maxima of activity display different freerunning periods ( τ a ≈ 23 h 50 min, τ b ≈ 22 h 15 min), suggesting the involvement of two oscillators controlling individually, the two activity peaks. Under continuous light, however, activity becomes unimodal, freerunning at τ ≈ 26 h 40 min. Resetting a 180° shift of high-low temperature cycles occurs faster than by light-dark cycles. In conditions of zeitgeber competition, by simultaneous exposition to light and temperature cycles at various phase relationships, light preponderantly controls the synchronization of activity. Results indicate that the activity of T. infestans is controlled by a circadian system involving a hierarchical arrangement of coupled oscillators. Environmental temperature modulates the activity level and the time course of the spontaneous locomotion. Antennectomy affects the temporal expression of the activity, insects becoming active at an unusual daytime.

Development of melatonin rhythm in the pineal gland and eyes of chick embryo.

A melatonin rhythm was observed in the pineals of 18-day-old chick embryos incubated under a light-dark regime of 18: 6 h. A low pineal melatonin content was found during the light phase of the day. Concentrations started to increase 2 h after dark onset and reached maximum levels after 4 h of darkness. The amplitude of the pineal melatonin rhythm increased considerably after 2 days and night-time concentrations in 20-day-old embryos were more than 5 times higher than in 18-day-old ones. Significant day/night differences in melatonin production were found both in pineals and eyes. Exposure of eggs to 1 h of light during the dark period decreased the high melatonin concentrations in the eyes but not in the pineals of the 20-day-old chick embryo. The results suggest that in this precocial bird at least part of the circadian system may already operate during embryonic life.

Influence of celestial light on visual and olfactory behavior of seed chalcids (Hymenoptera: Eurytomidae)

When the alfalfa [Bruchophagus roddi (Gussakovsky)], clover [Bruchophagus gibbus (Boheman)], and trefoil seed chaldds (TSC) [Bruchophagus platypterus (Walker)] were exposed to yellow, white, green, and purple painted polyethylene vials perforated by four small holes, only the latter species had a color preference, and that was for yellow, the color of its host flower. When TSC were exposed to green and yellow targets 5 h after sunrise, they preferred yellow targets but not 1 h after sunrise. The possibility of a circadian response was eliminated because different sequences of light-dark regimes prior to the test did not change the results. When TSC were exposed only to yellow targets, half of which had trefoil flowers hidden within, females preferred targets with flowers. When an identical test was conducted but with green instead of yellow targets, the preference for targets with flowers disappeared. In a four-choice test, TSC preferred yellow targets with or without flowers to green targets with or without flowers. Thus, TSC displayed an olfactory response only when the color yellow was present. In unfiltered skylight females preferred baited targets when the test began 3 h before or 1 h after solar noon but not 4 h before or 2 h after solar noon. Chalcids did display an olfactory preference 4 h before solar noon when a Polaroid filter was used to filter skylight and provide an east-west but not a north-south E-vector. When Helmholtz coils were used to apply a magnetic field that canceled or changed the direction of the earth's magnetic field, olfactory preference disappeared because the applied magnetic field changed TSC perception of the E-vector. In effect, TSC must perceive yellow in the presence of an east-west E-vector to display an olfactory preference to a choice of odors. We believe this is the first report that the E-vector of celestial light can influence olfactory and visual behavior of an insect.

In vitro growth response of Prunus cerasifera shoots as influenced by different light-dark cycles and sucrose concentrations

Trials were carried out to test if the higher growth response shown by shoot clusters of Mr. S. 2/5, a clonal selection of Prunus cerasifera, submitted to short and frequent light-dark regimes could be related to the amount of sucrose added to growth medium.

Transcriptional control of plastid gene expression during development of primary foliage leaves of barley grown under a daily light-dark regime

Plastid DNA transcription was studied during biogenesis and maturation of chloroplasts in primary foliage leaves of barley (Hordeum vulgare L.) seedlings grown under a daily light-dark regime. Specific age-dependent changes in the transcript pattern of plastids have been shown by hybridization to barley plastid DNA fragments of run-on transcript probes derived from homogeneous populations of proplastids and chloroplasts of different age. In proplastids, transcription of the rrn operon is predominant and the increasing transcriptional activity during biogenesis of chloroplasts is mainly the consequence of additional transcription of mRNA and tRNA genes. During maturation of chloroplasts, rrn transcription preferentially decreases and, conversely, transcription of the psbD gene increases. The importance of these changes in plastid DNA transcription is discussed in relation to chloroplast ageing.

Diazotrophic growth of the unicellular cyanobacterium Gloeothece sp. PCC 6909 in continuous culture

The unicellular cyanobacterium Gloeothece sp. PCC 6909 was grown diazotrophically in continuous culture. Various light-dark regimes and dilution rates were applied. Nitrogenase activity (acetylene reduction), oxygen-production and -consumption, and glycogen were measured over 24 h periods covering one complete light-dark cycle. Gloeothece fixed nitrogen predominantly in the light, concomitant with photosynthetic oxygen evolution. This result was found irrespective of the imposed light-dark regime or dilution rate. It is concluded that temporal separation of photosynthesis and nitrogen fixation is not obligatory in Gloeothece.

Effects of T cycles of light/darkness and periodic forced activity on methamphetamine-induced rhythms in intact and SCN-lesioned rats: Explanation by an hourglass-clock model

In this study intact and suprachiasmatic nuclei (SCN)-lesioned female rats were treated with chronic methamphetamine (MA) via the drinking water. Body temperature, feeding, drinking and wheel-running activity were continuously and automatically recorded. The rats were subjected to light-dark (LD) cycles with period T = 23 hr for 4 months and subsequently T = 25 hr for 3 months. Daily 3-hr forced activity (FA 3:21) was imposed during a few weeks under both LD regimes. MA induced infradian rhythms (period τ s = 28–54 hr) that were found to run parallel in all functions. In intact rats these infradian rhythms showed relative coordination by the LD regime and τ s shortened when T lengthened. In SCN-lesioned rats, however, the infradian rhythms were independent of the LD regime. Under the FA cycles τ s lengthening as well as synchronization was observed. We hypothesized that the MA-induced rhythms reflect a long-period sleep-wake cycle of the hourglass type. We investigated this hypothesis with a modified version of the hourglass-clock model of sleep regulation. Computer simulations showed that this model might offer an explanation for the experimental observations.

Control of olfactory-induced behavior in alfalfa seed chalcid (Hymenoptera: Eurytomidae) by celestial light

The behavioral response of female alfalfa seed chalcids is dependent on the polarization of sky light when exposed to olfactory stimuli between 1100 and 1500 hr. Females made no orientation flights to or landings on eight unbaited targets, but when half the targets were baited with the host-plant odor of hexyl acetate, they did make orientation flights to and landed on baited targets. Female preference for baited targets disappeared when natural sky light was plane polarized (at right angles to natural sky light polarization at 1200 hr). When natural sky light was passed through a diffuser filter to completely depolarize the light, females made numerous orientation flights but displayed no preference for baited targets. Any alteration of the natural wavelengths of sky light between 345 and 425 nm with various Kodak Wratten filters that excluded certain wavelengths produced similar results wherein females had no preference for baited or unbaited targets. The hypothesis that chalcid response was controlled by the polarization characteristics of sky light was confirmed when females again preferred baited targets in tests that were conducted 6 hr after sunrise, but they displayed no preference for baited targets when exposed to the mixture of polarized and nonpolarized light present in the sky 1-2 hr after sunrise or 1-2 hr before sunset. The possibility that chalcid response was due to a circadian rhythm was eliminated by exposure of insects to different sequences of light-dark regimes prior to the olfactory test.

Patterns of Pineal Melatonin Secretion in Rabbits: Diurnal Variation of Basal and Pulsatile Release

The level and release pattern of plasma melatonin in the confluens sinuum of 28 sighted and 18 blinded (i.e. acute bilateral orbital enucleation) rabbits anesthetized with pentobarbital were studied. The animals had been adapted to a 12:12 h light-dark regime. Blood samples were collected from the cannulated confluens sinuum and/or the femoral artery at either 2- or 4-min intervals in both the light and dark phases. Plasma melatonin was determined by radioimmunoassay. In all rabbits studied, plasma melatonin in the confluens sinuum exhibited an episodic release pattern, with pulses superimposed on a basal level. At 4-min sampling intervals, the pulsatile release of melatonin in sighted rabbits were 3.8 peaks/h in the light phase; shorter sampling interval (2-min) revealed more frequent pulsatile release of melatonin (5.1 peaks/h). In sighted animals in the light phase, the level of melatonin in the plasma of confluens sinuum was 7-15 times higher than that obtained from the plasma collected at the same time from the peripheral artery where the level of melatonin also exhibited pulsatile pattern. In blinded animals, melatonin levels in terms of mean concentration, mean maximum level and mean minimum (or baseline) level obtained in the dark phase were 12-14 times higher than those obtained in the light phase. These results suggest that the level of melatonin exhibits a diurnal rhythm in the confluens sinuum of rabbits.

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle.

The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied. In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed. Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only. Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.

Expression of Leaf Nitrate Reductase Genes from Tomato and Tobacco in Relation to Light-Dark Regimes and Nitrate Supply

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels.

Effects of intermittent lighting on sleep and activity in the domestic hen

This experiment deals with the effects of two different light-dark regimes on general activity, behavioural patterns and sleep in domestic hens ( Gallus domesticus). The light-dark schemes were composed of a traditional 14 h light-10h dark period and the Biomittent Lighting Program (BMLP), which consists of only 15 min light during every hour of the traditional 14 h photoperiod. Although it was found that hens are more active during the day than during the night, they were much less active under the BMLP-regime compared to the traditional regime. More specifically, there is considerable lowering of their activities during the dark portions of the day; behavioural observations revealed that they spend these periods not with resting behaviour, but with passive wakefulness. They more or less concentrate their daily activities during the 15-min light periods. Under the night period of the BMLP-regime, hens appeared to be more restless. Sleep recordings showed that sleep tended to decrease in favour of more drowsiness. It was concluded that circadian rhythmicity of the hens is maintained under both lighting schemes, although the BMLP-regime imposes more passive wakefulness during the day and slightly more activity during the night.

Carbohydrate formation in rewetted terrestrial cyanobacteria.

In the terrestrial cyanobacterium Nostoc commune Vauch. formation of carbohydrate polymers was measured upon rewetting the mats in a light-dark regime. To discriminate between carbohydrates of different physiological function, total carbohydrate was determined as anthrone-reactive material (ARM) and storage carbohydrate (glycogen) assayed by an enzymic test. In the dry thalli glycogen was found to represent less than one tenth of the ARM. After rewetting an increase of total carbohydrate was observed in illuminated samples. Only glycogen, however, showed a regular pattern of synthesis and degradation during a 12:12 h light-dark cycle. This indicates that most carbohydrates detected by anthrone belong to the metabolically inert sheath material.When illuminated colonies were kept submerged after rewetting glycogen was hydrolyzed indicative of being used in the rapid recovery of cellular functions as observed in rewetted colonies. Apparently, photosynthesis allowed for net glycogen synthesis only, provided the mats were sufficiently aerated. These findings give evidence that the (carbohydrate) sheath plays an important role in water retention in an organism bound to a terrestrial habitat.

Stage-Specific State I-State II Transitions during the Cell Cycle of Euglena gracilis

In synchronized Euglena gracilis (light-dark regime of 14:10 hours) the successive formation of the photosynthetic apparatus during cell ontogeny is correlated with large changes in photosynthetic efficiency (P Brandt, B von Kessel 1983 Plant Physiol 72: 616-619; B Kohnke, P Brandt 1984 Biochim Biophys Acta 766: 156-160). This observation led us to investigate the functional association of the chlorophyll a/b light-harvesting protein complex (LHCP) with photosystem I or II, because changes in energy flow to photosystem I or II and in energy transfer between the two photosystems can be a reason for these alterations. As criterion for the association of the LHCP with photosystem I or II, state transitions were determined after 15 minutes preillumination using wave-lengths of 725 or 620 nanometers. The state transitions were determined from measurements of fluorescence induction at room temperature, and fluorescence kinetics at 77 K. According to the obtained data (a) mobile LHCP is present only between the 6th and the 10th hour of the light-time of the cell cycle and (b) this functional relation of the LHCP to photosystem I only at this stage of Euglena chloroplast development is not accompanied by a decrease in stacking. A model for the organization of the newly inserted LHCP within the photosynthetic apparatus of E. gracilis is discussed.

Morphometric analysis of circadian variations in the retinal photoreceptor synaptic terminals of the adult and fetal guinea pig.

In order to test whether the alterations in photoreceptor synaptic terminal size and shape reported in lower vertebrates occur in a mammalian visual system, adult and fetal guinea pig retinas were exposed to an LD 12:12 lighting cycle, as well as to long-term light (LL) and long-term dark (DD) regimes. Representative random samples from all retinal quadrants, obtained at various times during these lighting regimes, were processed for electron microscopy. The synaptic terminals of all three photoreceptor cell types in this retina (alpha and paranuclear rods, and cones) were analyzed with computer-assisted morphometrics for changes in their area, perimeter, synaptic vesicle density, and the degree of plasmalemmal infolding. The data showed all three types of adult receptor terminals to have increased area and vesicle density, as well as decreased membrane infolding, during the light period, while both types of rods showed increased perimeter measurements in the dark. Results from adults maintained under extended lighting conditions (LL and DD) showed no difference when compared with sample times during a typical LD 12:12 lighting regimen where clear statistical differences existed. Data from fetal retinas showed no significant sustainable pattern in any of the measured variables. These quantitative findings have led to the conclusion that while alterations in perimeter measurements may be explained by using the vesicle recycling hypothesis, observed changes in terminal size and shape may be controlled by a light-initiated or light-enhanced mechanism and effected through an annular configuration of cross-striated fibrils found within these photoreceptor synaptic terminals.

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.

Entrainment of the circadian rhythm in rat pineal N-acetyltransferase activity under extremely long and short photoperiods.

Entrainment of a pacemaker driving the circadian rhythm in rat pineal N-acetyltransferase activity was studied under extremely long and short photoperiods. Adult male rats maintained under the light-dark regime (LD) 18:6 or under the regime LD 6:18 were exposed to a 1-min light pulse at different times at night, then they were released into darkness, and the next night phase-shifts of the evening N-acetyltransferase rise and of the morning N-acetyltransferase decline caused by light pulses were determined. The evening rise was phase-delayed by at most 0.5 h under LD 18:6, but by as much as 2.8 h under LD 6:18. The morning decline was phase-advanced by at most 1.9 h under LD 18:6, but by as much as 3.5 h under LD 6:18. Hence, the magnitude of phase-shifts and consequently patterns of phase-response curves, which show possibilities of discrete entrainment, depend on the photoperiods under which animals are maintained. A 1-min light pulse applied within 1 h before the end of the dark period phase-advanced the morning N-acetyltransferase decline under LD 18:6 as well as under LD 6:18, while a pulse applied within 1 h after the beginning of the dark period phase-delayed the evening N-acetyltransferase rise only in rats maintained under LD 18:6, but not in those kept under LD 6:18. It seems that under very long photoperiods, the N-acetyltransferase rhythm may be entrained by evening as well as by the morning light, while under very short photoperiods the rhythm may be synchronized by morning light only.

Light-dark regimes in the intertidal zone and tetrasporangial periodicity in the red algaRhodochorton purpureum

The causal factors for observed differences in tetrasporangial periodicity in high- and midlittoral plants in an estuarine population ofRhodochorton purpureum (Lightf.) Rosenv. were analysed. In high littoral plants tetrasporangia were absent from early March to early November when tetrasporogenesis was blocked by long days (>c. 12 h) and, from mid-May on also by high temperatures (>c. 16°C). Midlittoral plants, in particular those growing at shaded sites, continued to form tetrasporangia until the end of May. Here, at high tide, high water turbidity and heavy shading by brown algae reduced light to such an extent that it was below the threshold for light perception inR. purpureum for several hours. Since, on successive days, high water marks regularly fell c. 50 min later, very irregular light-dark regimes were the result. Days with two light and two dark periods (high tide at midday) are followed by days with one light and one, long, dark period (one high tide at dawn, the next at dusk). Experiments showed that these regimes are effectively short days forR. purpureum. In summer and autumn tetrasporogenesis in the midlittoral plants is blocked by high temperatures. Minimal daily temperature-values are often below the critical temperature, but this did not induce tetrasporangia. Probably average or maximal values block tetrasporogenesis and this was supported by experimental results. In one of the field samples a diminutive male gametophyte was found which had grown from a tetraspore that had germinated in situ, and this is the first report of a gametophyte ofR. purpureum from nature.