Circadian rhythms in plants: Strategies for analysis

Molecular mechanism of the circadian clock which regulates the circadian rhythms has been believed to be common in different organisms. However, recent topic about multiple oscillators in a cell is thought to suggest other possibility. We may need to reconsider effectiveness of strategies for understanding molecular mechanism of the circadian clock.     

Evidence for a plastic dual circadian rhythm in the oyster Crassostrea gigas

Although a significant body of literature has been devoted to the chronobiology of aquatic animals, how biological rhythms function in molluscan bivalves has been poorly studied. The first objective of this study was to determine whether an endogenous circadian rhythm does exist in the oyster, Crassostrea gigas. The second objective was to characterize it in terms of robustness. To answer these questions, the valve activity of 15 oysters was continuously recorded for 2 mo in the laboratory under different entrainment and free-running regimes using a high-frequency noninvasive valvometer. The present work demonstrates the presence of a circadian rhythm in the oyster Crassostrea gigas. First, oysters were entrained by 12 L:12 D conditions. Then, free-running conditions (D:D and L:L) indicated that the most frequently observed period ranged from 20 to 28 h, the circadian range. That endogenous circadian rhythm was characterized as weak. Indeed, the period (τ) of the individual animals exhibited high plasticity in D:D and L:L, and the animals immediately followed a 4-h phase advance or delay. Additionally, C. gigas appeared as a dual organism: all oysters were nocturnal at the beginning of the laboratory experiment (January), whereas they were diurnal at the end (March). That shift was progressive. Comparison with a full-year in situ record showed the same behavioral duality as observed in the laboratory: the animals were nocturnal in autumn-winter and diurnal in spring-summer. The significant advantage of a plastic and dual circadian rhythm in terms of adaptability in a highly changing environment is discussed.     

The oyster vasa-like gene: a specific marker of the germline in Crassostrea gigas

The vasa gene is a key determinant for germline formation in eukaryotes. This gene, highly conserved through evolution, encodes a RNA helicase protein member of the DEAD-box family. To understand the germline formation in oyster, we report here the isolation and the characterization of a vasa orthologue in Crassostrea gigas (Oyvlg). OyVLG contained the eight consensus domains of the DEAD-box including those providing RNA unwinding activity. The expression pattern of Oyvlg was examined in adult oyster tissues at different reproductive stages. Its expression was restricted to germline cells both in males and females, including germinal stem cells and auxiliary cells. The expression of Oyvlg, strongest in early germ cells, decreased as the maturation proceeded. These data and the evolutionary conservation observed suggested the role of Oyvlg in germline development. Oyvlg is the first germ cell specific marker in oyster and will be very useful in studies of oyster germline formation.     

Daily Expression of Six Clock Genes in Central and Peripheral Tissues of a Night-Migratory SongBird: Evidence for Tissue-Specific Circadian Timing

In birds, independent circadian clocks reside in the retina, pineal, and hypothalamus, which interact with each other and produce circadian time at the functional level. However, less is known of the molecular clockwork, and of the integration between central and peripheral clocks in birds. The present study investigated this, by monitoring the timed expression of five core clock genes (Per2. Cry1. Cry2. Bmal1, and Clock) and one clock-controlled gene (E4bp4) in a night-migratory songbird, the redheaded bunting (rb; Emberiza bruniceps). The authors first partially cloned these six genes, and then measured their 24-h profiles in central (retina, hypothalamus) and peripheral (liver, heart, stomach, gut, testes) tissues, collected at six times (zeitgeber time 2 [ZT2], ZT6, ZT11, ZT13, ZT18, and ZT23; ZT0?=?lights on) from birds (n?=?5 per ZT) on 12?h:12?h light-dark cycle. rbPer2. rbCry1. rbBmal1, and rbClock were expressed with a significant rhythm in all the tissues, except in the retina (only rbClock) and testes. rbCry2, however, had tissue-specific expression pattern: a significant rhythm in the hypothalamus, heart, and gut, but not in the retina, liver, stomach, and testes. rbE4bp4 had a significant mRNA rhythm in all the tissues, except retina. Further, rbPer2 mRNA peak was phase aligned with lights on, whereas rbCry1. rbBmal1, and rbE4bp4 mRNA peaks were phase aligned with lights off. rbCry2 and rbClock had tissue-specific scattered peaks. For example, both rbCry2 and rbClock peaks were close to rbCry1 and rbBmal1 peaks, respectively, in the hypothalamus, but not in other tissues. The results are consistent with the autoregulatory circadian feedback loop, and indicate a conserved tissue-level circadian time generation in buntings. Variable phase relationships between gene pairs forming positive and negative limbs of the feedback loop may suggest the tissue-specific contribution of individual core circadian genes in the circadian time generation.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

大鼠下丘脑交叉上核中CREB的昼夜节律

利用凝胶迁移率变化的实验方法,对饲养在光照－黑暗循环的条件和持续黑暗的条件下Wistar雄性大鼠下丘脑交叉上核中CREB含量的昼夜间变化进行了分析,发现CREB在交叉上核中具有内源性的昼夜节律.     

Population demography and genetic characteristics of the Pacific Oyster Crassostrea gigas in Japan

The Pacific oyster Crassostrea gigas is a commercially important bivalve distributed along the northwest Pacific coast. Here C. gigas in Japan was investigated using mtDNA and microsatellite markers to elucidate its genetic structure and phylogeny. On the basis of mtDNA all populations showed high genetic diversity with limited genetic differentiation among populations. The pattern of MtDNA diversity suggested that C. gigas had experienced population expansion about 112 Kya, prior to the last glacial maximum (LGM), which accorded well with other marine organisms. For microsatellites, a Bayesian-based assignment test demonstrated that C. gigas is nearly panmictic. However, on the basis of estimates of F_ST, Kumano populations differed significantly from other populations, a recent occurrence based on low R_ST. Irrespective of geographical distance, genetic similarity was observed in the main aquaculture regions with large-scale transportation of cultured spat. Unlike in the Yellow Sea, a genetic bottleneck was not detected in Japanese populations. These results imply, contrary to the prevailing view, that C. gigas in Japan was demographically stable during the LGM. Gene flow by larval dispersal seems to be regionally restricted to localities of congenital areas by ocean currents, while genetic homogenization by cultivated oysters might have occurred in aquaculture areas.     

Use of metallothionein in gills from oysters (Crassostrea gigas) as a biomarker: seasonal and intersite fluctuations

The use of oyster gills for the analytical determination of metallothionein (MT) concentration as a biomarker of metal exposure was investigated. Temporal variations in MT and metal concentrations (which can interfere with inter-site differences) were examined over a 7 month period (from spring to autumn) in Japanese oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site (Gironde estuary, France) as well as in individuals translocated from the clean to the contaminated area. The ratio between the annual average of MT concentrations in specimens from the clean and the metal-rich sites was 1.3. During the last 3 months of the experiment, significant differences were no longer registered between transplants and residents from the Gironde estuary. Metals concentrations in oyster gills differed consistently between the clean and the metal-rich sites (annual average ratios of 1.5, 2.7 and 9.8, respectively, for zinc, copper and cadmium) and a fast increase in metal concentrations (over a few months) was observed in transplants, mainly for cadmium. MT and soluble metal concentrations were found to be positively and significantly correlated over the period of the study. This relationship is a positive argument for a possible use of gill MT concentration as a biomarker of metal pollution in contrast to previous findings on the digestive gland, there being a smaller amount of seasonal variability in the weight of oyster gills.     

蓝藻生物节律性分子调控机制的研究进展

生物钟现象是一种普遍存在于生物界细胞的内源节律性保持机制。生物钟机制的存在可以使生物体的代谢行为产生并维持以24 h为周期的昼夜节律,从而更好地适应于地球自转所产生的环境条件昼夜间节律性变化。蓝藻是目前生物钟分子机制研究中的模式生物,其依赖于k ai基因家族成员的核心生物钟调控模式已经被众多研究者详细阐明。蓝藻生物钟的核心振荡器是由蓝藻k aiA/B/C的编码产物来调控的,Kai蛋白的表达模式具有节律性。KaiC蛋白磷酸化状态的节律性循环及输入、输出途径相关组成蛋白的翻译后修饰状态节律性循环共同组成其反馈回路,负责维持生物钟节律性振荡的持续进行并与环境周期保持同步。传统的蓝藻生物钟分子机制模型认为,节律性表达基因翻译产物的转录/翻译负反馈抑制环是生物节律性维持和输出的关键。遗憾的是,在其它物种生物钟分子机制研究中未发现由kai基因家族成员同源基因组成的节律性标签,这表明以k aiA/B/C为核心振荡器的生物钟系统并不是一种跨物种保守的生物钟系统。近期,人们发现非转录/翻译依赖的振荡器(NTO)也具有成为生物节律性产生和维持的“源动力”的可能。过氧化物氧化还原酶(PRX)氧化还原状态节律性是第一种被报道的跨物种保守的NTO节律性标签,这也日渐成为蓝藻生物钟分子机制研究新的热点。     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

Cerebral Cortical Glucose Utilization in the Conscious Rat: Evidence for a Circadian Rhythm

Abstract: The presence of a circadian rhythm of glucose utilization was demonstrated in vivo in rat cerebral cortex. The activity pattern of the rats, living in a controlled lighting regimen with lights on from 7 a.m. to 7 p. m., appeared to coincide with the rate of glucose consumption in the brain. The rate of utilization was measured at 3-h intervals throughout the day and was found to fall from a maximum at 3 a.m. of 0.98 ± 0.13 μmol min⁻¹ g⁻¹ to a minimum of 0.70 ± 0.08 μmol min⁻¹ g⁻¹ at 3 p. m. Brain glucose also varied with time and its fluctuating level weakly correlated with its rate of utilization. Animals entrained on a 5-h (4: 30-9: 30 p. m.) feeding schedule had a similar circadian rhythm, with only a slight increase in amplitude. Reversal of the light cycle caused a disruption in the normal rhythm, but utilization still varied significantly with time of day. The results both indicate the potential error that can be encountered in experiments done at different times of the day and stress the need for awareness of time of day as a factor in measurements of alterations of metabolic rate in the brain.     

Endogenous Circadian Rhythm is a Crucial Tool for Survival of the Sand-desert Tenebrionid Beetle Trigonoscelis gigas Reitter

The beetle T. gigas is interesting as a successful survivor among competing species in a hostile, arid, environment (the sand-desert), and as a promising biological model system in laboratory experiments. Behaviour and motor activity rhythms of T. gigas were studied in the field, using autonomous monitors. In order to avoid extreme temperatures, this beetle developed a peculiar behavioural strategy: narrow time windows for activity (in the morning and in the evening). The rest of the time it spends deep in the sand, at constant comfortable temperature and in continuous darkness, in temporal isolation. We demonstrated that there are no available Zeitgebers for the beetle during its rest interval. Hence, a reliable circadian clock is the only way for T. gigas to implement the proper timing of its activity. It means, that the circadian timing system of this beetle is not just a source of relative advantage, but is a crucial prerequisite of survival.     

Endogenous Circadian Rhythm is a Crucial Tool for Survival of the Sand-desert Tenebrionid Beetle Trigonoscelis gigas Reitter

The beetle T. gigas is interesting as a successful survivor among competing species in a hostile, arid, environment (the sand-desert), and as a promising biological model system in laboratory experiments. Behaviour and motor activity rhythms of T. gigas were studied in the field, using autonomous monitors. In order to avoid extreme temperatures, this beetle developed a peculiar behavioural strategy: narrow time windows for activity (in the morning and in the evening). The rest of the time it spends deep in the sand, at constant comfortable temperature and in continuous darkness, in temporal isolation. We demonstrated that there are no available Zeitgebers for the beetle during its rest interval. Hence, a reliable circadian clock is the only way for T. gigas to implement the proper timing of its activity. It means, that the circadian timing system of this beetle is not just a source of relative advantage, but is a crucial prerequisite of survival.