Differential Involvement of the Circadian Clock in the Expression of Genes Required for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Synthesis,Assembly, and Activation in Arabidopsis thaliana

We have investigated the role of the circadian clock in the regulation of expression of genes required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) synthesis, assembly, and activation. Circadian oscillations in RCA (the gene encoding Rubisco activase) and RBCS (the gene encoding Rubisco small subunit) mRNA accumulation, with peak abundance occurring soon after dawn, occur in Arabidopsis thaliana grown in a light-dark (LD) photoperiod. These oscillations persist in plants that have been transferred from LD to either continuous darkness (DD) or continuous light (LL). In contrast, CPN60[alpha] (the gene encoding [alpha]-chaperonin) and CPN60[beta] (the gene encoding [beta]-chaperonin) mRNA abundance oscillates in a diurnal, but not in a circadian, fashion. Although rapid damping of the circadian oscillation in RCA mRNA abundance is observed in Arabidopsis that have been grown in LD and then transferred to DD for 2 d, the circadian oscillations in RCA and RBCS mRNA abundance persist for at least five continuous cycles in LL, demonstrating the robustness of the circadian oscillator.     

Interactions between Light and the Circadian Clock in the Regulation of CAT2 Expression in Arabidopsis

In Arabidopsis seedlings germinated and grown in continuous light, CAT2 mRNA abundance peaks 1 d after imbibition, consistent with the role of catalase in detoxifying H2O2 generated during the [beta]-oxidation of fatty acids stored in the seed. A second peak of CAT2 mRNA abundance, of lower amplitude than the initial peak, appears 6 d after imbibition and may be associated with the development of photosynthetic competence and induction of photorespiration. This second peak in steady-state CAT2 mRNA abundance is regulated by light and is not seen in etiolated seedlings. CAT2 mRNA accumulation is induced by exposure to high-fluence blue or far-red light but not by red light. In addition, light induction is unaffected by several mutations that block blue light-mediated inhibition of hypocotyl elongation (blu1, blu2, blu3, hy4), suggesting phytochrome involvement. When etiolated seedlings are transferred to continuous white light, CAT2 mRNA rapidly (within 30 min) accumulates. It is interesting that in these seedlings CAT2 mRNA abundance undergoes pronounced oscillations with a circadian (24 h) periodicity, indicating control by the endogenous circadian clock. No such oscillations are detected in CAT2 mRNA abundance in etiolated seedlings prior to illumination. Control of CAT2 expression by the circadian clock is also seen in 5-week-old plants grown in a light-dark cycle and transferred either to continuous dark or to continuous light; in continuous light the circadian oscillations in CAT2 mRNA abundance persist for at least five circadian cycles, indicating the robustness of this circadian rhythm.     

水稻叶片Rubisco 活化酶表达的昼夜变化

在 48h的自然光照、连续光照和连续黑暗处理下 ,水稻幼苗rcamRNA的含量均表现出昼夜节奏特性 ,其中以自然光周期中变化最为明显。在光暗交替的条件下 ,RCA含量虽也表现出明显的昼夜变化 ,但连续黑暗处理其含量持续下降 ,连续光照则其含量先上升然后下降 ,说明它不发生节昼现象。这些结果表明RCA表达在转录和翻译水平上的调控机制不同 ,转录既由光暗交替控制又受内生节奏调节 ,而翻译则更大程度上由光调节     

Oscillations in the Activities of Enzymes of Nitrate Reduction and Ammonia Assimilation in Glycine max and Zea mays

The activities of glutamate dehydrogenase (GDH), glutamine synthetase (GS), and nitrate reductase (NR) and the levels of soluble protein and NO^-₃ were assayed in soybean (Glycine max [L.] Merr.) leaves over a 48-h period with the initial 24 h under a light-dark cycle (LD 16:8) followed by 24 h of continuous light (LL). Plants had been entrained for 30 days under the LD regime. Maize (Zea mays) leaves (10 days old) under a LD 15:9 cycle were assayed only for NR and nitrite reductase (NiR). Data were subjected to frequency analysis by the least squares method to determine probabilities for cosine function periods (τ's) between 10 and 30 h. NR activities for both soybean and Zea leaves had 24 h τ's with P values < 0.05 indicating circadian periodicity. GDH in soybeans had a 24-h rhythm under LD conditions which lengthened under LL conditions. The 24-h rhythm of GDH displayed maximal activity toward the end of the dark period of the LD cycle whereas the highest activity of NR was early in the light period. Total soluble protein displayed a rhythm with a best fitting τ of greater than 24 h under both LD and LL. GDH, GS, NR, NO₃, and soluble protein in soybeans and NiR in Zea, all displayed that were ultradian (10–18 h), indicating that a τ of about one half a circadian periodicity may be a common characteristic of the enzymes of primary nitrogen metabolism in higher plants. These data also demonstrate that although both NR and GDH are circadian in their activity, the 24-h rhythm may be greatly influenced by ultradian oscillations in activity.     

Developmental plasticity of the locomotor activity rhythm of Drosophila melanogaster

We used four replicate outbred populations of Drosophila melanogaster to investigate whether the light regimes experienced during the pre-adult (larval and pupal) and early adult stages influence the free-running period (τ_DD) of the circadian locomotor activity rhythm of adult flies. In a series of two experiments four different populations of flies were raised from egg to eclosion in constant light (LL), in light/dark (LD) 12:12 h cycle, and in constant darkness (DD). In the first experiment the adult male and female flies were directly transferred into DD and their locomotor activity was monitored, while in the second experiment the locomotor activity of the emerging adult flies was first assayed in LD 12:12 h for 15 days and then in DD for another 15 days. The τ_DD of the locomotor activity rhythm of flies that were raised in all the three light regimes, LL, LD 12:12 h and in DD was significantly different from each other. The τ_DD of the locomotor activity rhythm of the flies, which were raised in DD during their pre-adult stages, was significantly shorter than that of flies that were raised as pre-adults in LL regime, which in turn was significantly shorter than that of flies raised in LD 12:12 h regime. This pattern was consistent across both the experiments. The results of our experiments serve to emphasise the fact that in order to draw meaningful inferences about circadian rhythm parameters in insects, adequate attention should be paid to control and specify the environment in which pre-adult rearing takes place. The pattern of pre-adult and early adult light regime effects that we see differs from that previously observed in studies of mutant strains of D. melanogaster, and therefore, also points to the potential importance of inter-strain differences in the response of circadian organisation to external influences.     

Light- and temperature-entrained circadian regulation of activity and mRNA accumulation of 1-aminocyclopropane-1-carboxylic acid oxidase in Stellaria longipes

Stem and leaf tissues of Stellaria longipes Goldie (prairie ecotype) exhibit circadian rhythmicity in the activity and mRNA abundance for 1-aminocyclopropane-1-carboxylic acid oxidase (EC 1.4.3). The steady-state mRNA levels and enzymatic activity levels fluctuated with a period of approximately 24 h and reached their maxima by the middle of the light phase and minima by the middle of the dark phase. The oscillations showed damping under constant light, constant dark and constant temperature conditions, indicating that the rhythm is entrained by an external signal. The results indicate that light/dark cycles have greater entraining effects than temperature cycles. A 15-min red light pulse, but not a blue light pulse, could reset rhythm in continuous darkness, suggesting the possible role of a red-light signal transduction pathway in the circadian regulation of 1-aminocyclopropane-1-carboxylic acid oxidase.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DD continuous dark - LD light-dark - LL continuous light - ZT Zeitgeber time (start of light period for circadian entrainment) This study was supported by operating grants to C.C.C., and D.M.R. from the Natural Sciences and Engineering Research Council of Canada.The authors gratefully acknowledge the award of a Bettina Bahlsen memorial Graduate Scholarship by University of Calgary to A.K. We are grateful to Dr. M.M. Moloney for allowing the use of his laboratory facilities.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

EFFECT OF LIGHT ON THE DEVELOPMENT OF THE CIRCADIAN RHYTHM OF MOTOR ACTIVITY IN THE MOUSE

In previous experiments, we found that rats raised in constant light (LL) manifested a more robust circadian rhythm of motor activity in LL and showed longer phase shifts after a light pulse in constant darkness (DD) than those raised under constant darkness. In addition, we observed that the effects produced by constant light differed depending on the time of postnatal development in which it was given. These results suggest that both sensitivity to light and the functioning of the circadian pacemaker of the rat could be affected by the environmental conditions experienced during postembryonic development. Thus, the present experiment aimed to study whether postnatal exposure to light could also affect the circadian system of the mouse. Three groups of mice were formed: One group was raised under constant darkness during lactation (DD group), the second under constant light (LL group), and the third under light-dark cycles (LD group). After lactation, the three groups were submitted first to constant light of high intensity, then to LD cycles, and finally to constant darkness. In the DD stage, a light pulse was given. Finally, mice were submitted to constant light of low intensity. We observed that the circadian rhythm of the DD group was more disturbed under constant light than the rhythm of the LL group, and that, when light intensity increased, the period of the rhythm of the DD group lengthened more than that of the LL group. No significant differences among the groups were found in the phase shift induced by the light pulse. Therefore, it appears that DD mice are more sensitive to light than their LL counterparts. However, at present there is no evidence to affirm that the light environment experienced by the mouse during postnatal development affects the circadian pacemaker. (Chronobiology International, 18(4), 683–696, 2001)     

Locomotor activity rhythm in two wrasses,Halichoeres tenuispinnis andPteragogus flagellifera,under various light conditions

The locomotor activity rhythms were examined by using an actograph with infra-red photo-electric switches for two species of wrasses, (Halichoeres tenuispinnis andPteragogus flagellifera) under various light conditions. InH. tenuispinnis, the locomotor activity of almost all fish under light-dark cycle regimen (LD12:12; 06:00–18:00 light, 18:00–06:00 dark) commenced somewhat earlier than the beginning of light period and continued till somewhat earlier than the beginning of the dark period. This species clearly showed free-running activity rhythms under both constant illumination (LL) and constant darkness (DD). Therefore,H. tenuispinnis appeared to have a circadian rhythm. The length of the circadian period ranged from 23 hr. 30 min. to 23 hr. 44 min. under LL, and was from 23 hr. 39 min. to 24 hr. 18 min. under DD. On the other hand, the locomotor activity ofP. flagellifera occurred mostly in the light period under LD 12:12. The activity of this species continued through LL, but was greatly suppressed in DD, so that none of the fish had any activity rhythm in both constant conditions. It was known from field observations thatH. tenuispinnis burrowed and lay in sandy bottoms, whileP. flagellifera hid and rested in bases of seagrasses and shallow crevices of rocks during the night. In the present two wrasses, it seemed that the above-mentioned difference of noctural behavior was closely related to the intensity of the endogenous factor in the activity rhythm.     

Turkey retina and pineal gland differentially respond to constant environment

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.     

Unterschiedliche Lichtintensitätsabhängigkeit der Spontanperioden als Ursache interner Desynchronisation circadianer Rhythmen bei Phaseolus coccineus

Summary The length of the free-running periods of circadian leaf movements in the primary and secondary pulvini of Phaseolus coccineus was measured in constant darkness (DD) and continuous light (LL) of different intensities. The periods for the two pulvini do not differ from each other in DD and LL 30000 lx. However, they differ in LL 60 lx, 600 lx and 6000 lx, indicating a state of internal desynchronization. These results show a difference in the light intensity dependence of the 2 oscillations and a lack of mutual synchronization between them.     

Differential Regulation of Arylalkylamine N-Acetyltransferase Activity in Chicken Retinal Ganglion Cells by Light and Circadian Clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30–60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar)     

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO₃ (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.     

Circadian rhythmicity in the expression of a novel light-regulated rice gene

We have identified and analyzed cDNAs corresponding to a single-copy gene from rice, designated lir1, whose expression exhibits dramatic diurnal fluctuations. The cDNAs encode a putative protein of 128 amino acids with no homology to known proteins. Lir1 mRNA accumulates in the light, reaching maximum and minimum steady-state levels at the end of the light and dark period, respectively. The oscillations of lir1 mRNA abundance persist after the plants have been transferred to continuous light or darkness. Plants germinated in the dark have very low levels of lir1 mRNA, whereas plants germinated in continuous light express lir1 at an intermediate but constant level. These results indicate that lir1 expression is controlled by light and a circadian clock.