Endogenous Timekeeping Mechanisms Research Articles

Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology.

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

B-Box proteins BBX28 and BBX29 interplay with PSEUDO-RESPONSE REGULATORS to fine-tune circadian clock in Arabidopsis.

As an endogenous time-keeping mechanism, the circadian clock benefits plant fitness and adaptation to the rhythmically changed diel environments. The key components within the core oscillator of plant circadian clock have been extensively characterised, however, the fine-tuning circadian regulators are still less identified. Here, we demonstrated that BBX28 and BBX29, the two B-Box V subfamily members lacking DNA-binding motifs, are involved in the regulation of Arabidopsis circadian clock. Over-expressing either BBX28 or BBX29 significantly lengthened circadian period, whereas loss-of-function of BBX28 rather than BBX29 displayed a modestly long period in free-running condition. Mechanistically, BBX28 and BBX29 interacted with core clock components PRR5, PRR7 and PRR9 in nucleus to augment their transcriptional repressive activities. RNA-sequencing analysis further revealed that BBX28 and BBX29 shared 686 common differentially expressed genes (DEGs) including a subset of known direct transcriptional targets of PRR proteins within core oscillator, including CCA1, LHY, LNKs and RVE8 etc. Intriguingly, PRR proteins can feedback repress BBX28 and BBX29 transcription by associating with their promoters. Together, our findings unmasked an exquisite mechanism in which BBX28 and BBX29 interplay with PRR proteins to fine-tune the circadian pace.

Impact of disabled circadian clock on yellow fever mosquito Aedes aegypti fitness and behaviors

Like other insects, Aedes aegypti displays strong daily patterns in host seeking and mating. Much of these behaviors are believed to be under the control of a circadian clock, an endogenous timekeeping mechanism relying on transcriptional/translational negative feedback loops that drive rhythmic physiology and behavior. To examine the connection between the circadian clock and various Ae. aegypti behaviors, we knocked out the core clock gene cycle using CRISPR/Cas9. We found that the rhythmic pattern and intensity of mRNA expression of seven circadian genes, including AeCyc−/−, were altered across the day/night cycle as well as in constant darkness conditions. We further show that the mutant CYC protein is incapable of forming a dimer with CLK to stimulate per expression and that the endogenous clock is disabled in AeCyc−/− mosquitoes. AeCyc−/− do not display the bimodal locomotor activity pattern of wild type, have a significantly reduced response to host odor, reduced egg hatching rates, delayed embryonic development and reduced adult survival and mating success. Surprisingly however, the propensity to blood feed in AeCyc−/− females is significantly higher than in wildtype females. Together with other recent work on the circadian clock control of key aspects of mosquito biology, our data on how cycle KO affects mosquito behavior and fitness provides a basis for further work into the pathways that connect the mosquito endogenous clock to its vector competence.

Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor.

Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night. The molecular basis for such temperature-dependent behavioural modulation has been associated with core circadian clock genes, but the neuronal basis is not yet clear. Under relatively cool temperatures such as 25°C, the role of the circadian pacemaker ventrolateral neurons (LNvs), along with a major neuropeptide secreted by them, pigment dispersing factor (PDF), has been showed in regulating various aspects of locomotor activity rhythms. However, the role of the LNvs and PDF in warm temperature-mediated behavioural modulation has not been explored. We show here that flies lacking proper PDF signalling or the LNvs altogether, cannot suppress their locomotor activity resulting in loss of sleep during the middle of the night, and thus describe a novel role for PDF signalling and the LNvs in behavioural modulation under warm ambient conditions. In a rapidly warming world, such behavioural plasticity may enable organisms to respond to harsh temperatures in the environment.

Daily Regulation of Key Metabolic Pathways in Two Seagrasses Under Natural Light Conditions

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external environmental cycles. It produces rhythms of plant metabolism and physiology, and interacts with signaling pathways controlling daily and seasonal environmental responses through gene expression regulation. Downstream metabolic outputs, such as photosynthesis and sugar metabolism, besides being affected by the clock, can also contribute to the circadian timing itself. In marine plants, studies of circadian rhythms are still way behind in respect to terrestrial species, which strongly limits the understanding of how they coordinate their physiology and energetic metabolism with environmental signals at sea. Here, we provided a first description of daily timing of key core clock components and clock output pathways in two seagrass species, Cymodocea nodosa and Zostera marina (order Alismatales), co-occurring at the same geographic location, thus exposed to identical natural variations in photoperiod. Large differences were observed between species in the daily timing of accumulation of transcripts related to key metabolic pathways, such as photosynthesis and sucrose synthesis/transport, highlighting the importance of intrinsic biological, and likely ecological attributes of the species in determining the periodicity of functions. The two species exhibited a differential sensitivity to light-to-dark and dark-to-light transition times and could adopt different growth timing based on a differential strategy of resource allocation and mobilization throughout the day, possibly coordinated by the circadian clock. This behavior could potentially derive from divergent evolutionary adaptations of the species to their bio-geographical range of distributions.

An HSP90 co-chaperone controls circadian proteostasis

The circadian clock is an endogenous timekeeping mechanism that can sustain oscillations in gene expression, metabolism and other aspects of physiology with a near 24 hour periodicity. Circadian sy...

Circadian rhythms of retinal sensitivity in the Atlantic tarpon, Megalops atlanticus

The Atlantic tarpon (Megalops atlanticus Valenciennes,1847) is a visually-guided marine predator that may be active at any time in the 24-hr light-dark cycle despite dramatic changes in light intensity over the course of the day. To test the hypothesis that retinal sensitivity changes with time of day in M. atlanticus, possibly under the influence of a circadian clock, populations of fish (three populations of 6 fish per lighting treatment for two treatments = 36 fish total) were exposed to different lighting treatments and sensitivity of the retina was measured periodically by electroretinography (ERG). The intensity of light required to elicit a half-maximal ERG response was significantly greater during the day than the night in fish held in 12L:12D light-dark cycles (LD). To determine whether this cycle of retinal sensitivity is driven by an internal timekeeping mechanism, ERG was performed at 4-hr intervals over the course of 24 hrs in constant darkness (DD). Sensitivity was significantly higher during subjective night than during subjective day, though the rhythm in DD was damped relative to the cycle in LD. These results show that retinal sensitivity is much higher at night than during the day in a light-dark cycle, and that this cycle of retinal sensitivity is driven at least in part by an internal biological clock. Such endogenous timekeeping mechanisms enhance survivability by allowing organisms to anticipate change in their external environments. Rhythms of retinal sensitivity are likely important for survival by supporting prey capture, predator avoidance, and reproduction, but they may be disrupted by abnormally-timed exposure to light, including artificial light at night.

Diminished circadian rhythms in hippocampal microglia may contribute to age-related neuroinflammatory sensitization

Aged animals exhibit diminished circadian rhythms, and both aging and circadian disruption sensitize neuroinflammatory responses. Microglia—the innate immune cell of the central nervous system—possess endogenous timekeeping mechanisms that regulate immune responses. Here, we explored whether aging is associated with disrupted diurnal rhythms in microglia and neuroinflammatory processes. First, hippocampal microglia isolated from young rats (4 months F344XBN) rhythmically expressed circadian clock genes, whereas microglia isolated from the hippocampus of aged rats (25 months) had aberrant Per1 and Per2 rhythms. Unstimulated microglia from young rats exhibited robust rhythms of TNFα and IL-1β mRNA expression, whereas those from aged rats had flattened and tonically elevated cytokine expression. Similarly, microglial activation markers were diurnally regulated in the hippocampus of young but not aged rats and diurnal differences in responsiveness to both ex vivo and in vivo inflammatory challenges were abolished in aged rats. Corticosterone is an entraining signal for extra–suprachiasmatic nucleus circadian rhythms. Here, corticosterone stimulation elicited similar Per1 induction in aged and young microglia. Overall, these results indicate that aging dysregulates circadian regulation of neuroinflammatory functions.

Diminished circadian rhythms in microglia may contribute to age-related neuroinflammatory sensitization

Circadian rhythms change dramatically across the lifespan, with aged animals exhibiting diminished molecular and behavioral circadian rhythms. Both aging and circadian disruption are associated with sensitized neuroinflammatory processes. We previously demonstrated that microglia – the primary innate immune cell of the central nervous system – possess endogenous circadian timekeeping mechanisms that regulate immune responses. Here, we explored whether age-associated neuroinflammatory sensitization is associated with disrupted diurnal rhythms in microglia. Hippocampal microglia were isolated from aged (24 mos.) and young FBN rats at 6 h intervals. As expected, microglia from young rats rhythmically expressed core circadian clock genes. Microglia from aged rats displayed comparable rhythms in several clock genes including BMAL1 and Rev-erb; however, rhythmic expression of Per1 and Per2 were completely ablated. Unstimulated microglia from young rats also exhibited robust rhythms of TNFalpha and IL1beta mRNA, with peak cytokine expression during the middle of the light phase. In contrast, aged microglia had tonically elevated inflammatory cytokines that were not regulated by time-of-day. Similarly, diurnal differences in responsivity to ex vivo stimulation with lipopolysaccharide were not apparent in aged microglia. Corticosterone treatment induced Per1 expression in aged and young microglia; however, glucocorticoid rhythms were suppressed in the aged hippocampus. This suggests that changes in entrainment signals may be disrupted with aging. Overall, here we show intrinsic microglial rhythms in clock and inflammatory genes are dysregulated with aging.

Vision and photoentrainment in fishes: the effects of natural and anthropogenic perturbation.

Vision and photoentrainment in fishes are vital for feeding, avoiding predation, spatial orientation, navigation, social communication and the synchronization of many homeostatic functions such as activity patterns and sleep. The camera-like (image-forming) eyes of fishes are optimized to provide a clear view of their preferred ecological niche, while non-visual photoreceptors provide irradiance detection that mediates circadian photoentrainment, an endogenous time-keeping mechanism (biological clock) to respond to predictable changes in environmental conditions. Fish and fisheries are under pressure from both natural and anthropogenic perturbation, which in many cases alters the intensity and spectral composition of the light environment on which they depend for their survival. This review examines the effects of a changing light environment and turbidity on the health of fishes within a developmental and ecological context. Understanding the sensory environment of fishes is vital to predicting their responses and, ultimately, their resilience to environmental change and the potential for maintaining sustainable levels of biodiversity.

Animal clocks: when science meets nature

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian 'clock'), with the alternation of environmental light and darkness synchronizing (entraining) these rhythms to the natural day-night cycle. Our knowledge of the circadian system of animals at the molecular, cellular, tissue and organismal levels is remarkable, and we are beginning to understand how each of these levels contributes to the emergent properties and increased complexity of the system as a whole. For the most part, these analyses have been carried out using model organisms in standard laboratory housing, but to begin to understand the adaptive significance of the clock, we must expand our scope to study diverse animal species from different taxonomic groups, showing diverse activity patterns, in their natural environments. The seven papers in this Special Feature of Proceedings of the Royal Society B take on this challenge, reviewing the influences of moonlight, latitudinal clines, evolutionary history, social interactions, specialized temporal niches, annual variation and recently appreciated post-transcriptional molecular mechanisms. The papers emphasize that the complexity and diversity of the natural world represent a powerful experimental resource.

Socially synchronized circadian oscillators.

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian 'clock'). The alternation of environmental light and darkness synchronizes (entrains) these rhythms to the natural day-night cycle, and underlying mechanisms have been investigated using singly housed animals in the laboratory. But, most species ordinarily would not live out their lives in such seclusion; in their natural habitats, they interact with other individuals, and some live in colonies with highly developed social structures requiring temporal synchronization. Social cues may thus be critical to the adaptive function of the circadian system, but elucidating their role and the responsible mechanisms has proven elusive. Here, we highlight three model systems that are now being applied to understanding the biology of socially synchronized circadian oscillators: the fruitfly, with its powerful array of molecular genetic tools; the honeybee, with its complex natural society and clear division of labour; and, at a different level of biological organization, the rodent suprachiasmatic nucleus, site of the brain's circadian clock, with its network of mutually coupled single-cell oscillators. Analyses at the 'group' level of circadian organization will likely generate a more complex, but ultimately more comprehensive, view of clocks and rhythms and their contribution to fitness in nature.

Transcriptional repressor PRR5 directly regulates clock-output pathways

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.

Retinoic acid receptors move in time with the clock in the hippocampus. Effect of a vitamin-A-deficient diet

An endogenous time-keeping mechanism controls circadian biological rhythms in mammals. Previously, we showed that vitamin A deficiency modifies clock BMAL1 and PER1 as well as BDNF and neurogranin daily rhythmicity in the rat hippocampus when animals are maintained under 12-h-light:12-h-dark conditions. Retinoic acid nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been detected in the same brain area. Our objectives were (a) to analyze whether RARα, RARβ and RXRβ exhibit a circadian variation in the rat hippocampus and (b) to investigate the effect of a vitamin-A-deficient diet on the circadian expression of BMAL1, PER1 and retinoic acid receptors (RARs and RXRβ) genes. Holtzman male rats from control and vitamin-A-deficient groups were maintained under 12-h-light:12-h-dark or 12-h-dark:12-h-dark conditions during the last week of treatment. RARα, RARβ, RXRβ, BMAL1 and PER1 transcript and protein levels were determined in hippocampus samples isolated every 4 h in a 24-h period. Regulatory regions of RARs and RXRβ genes were scanned for clock-responsive sites, while BMAL1 and PER1 promoters were analyzed for retinoic acid responsive elements and retinoid X responsive elements. E-box and retinoid-related orphan receptor responsive element sites were found on regulatory regions of retinoid receptors genes, which display an endogenously controlled circadian expression in the rat hippocampus. Those temporal profiles were modified when animals were fed with a vitamin-A-deficient diet. Similarly, the nutritional vitamin A deficiency phase shifted BMAL1 and abolished PER1 circadian expression at both mRNA and protein levels. Our data suggest that vitamin A deficiency may affect the circadian expression in the hippocampus by modifying the rhythmic profiles of retinoic acid receptors.

Circadian oscillation of gibberellin signaling in Arabidopsis

Circadian clocks are endogenous timekeeping mechanisms that allow organisms to anticipate rhythmic, daily environmental changes. Temporal coordination of transcription results in a set of gene expression patterns with peak levels occurring at precise times of the day. An intriguing question is how a single clock can generate different oscillatory rhythms, and it has been proposed that hormone signaling might act in plants as a relay mechanism to modulate the amplitude and the phase of output rhythms. Here we show that the circadian clock gates gibberellin (GA) signaling through transcriptional regulation of the GA receptors, resulting in higher stability of DELLA proteins during daytime and higher GA sensitivity at night. Oscillation of GA signaling appears to be particularly critical for rhythmic growth, given that constitutive expression of the GA receptor expands the daily growth period in seedlings, and complete loss of DELLA function causes continuous, arrhythmic hypocotyl growth. Moreover, transcriptomic analysis of a pentuple della KO mutant indicates that the GA pathway mediates the rhythmic expression of many clock-regulated genes related to biotic and abiotic stress responses and cell wall modification. Thus, gating of GA sensitivity by the circadian clock represents an additional layer of regulation that might provide extra robustness to the diurnal growth rhythm and constitute a regulatory module that coordinates the circadian clock with additional endogenous and environmental signals.

A CRY FROM THE KRILL

Antarctic krill (Euphausia superba) inhabit a region with strong seasonality in several parameters, such as photoperiod, light intensity, extent of sea ice, and food availability. In particular, seasonal changes in environmental light regimes have been shown to strongly influence krill metabolism, representing control signals for seasonal regulation of physiology of this key Southern Ocean species. Here, we report the identification of a cryptochrome gene, a cardinal component of the clockwork machinery in several organisms. EsCRY appears to be an ortholog of mammalian-like CRYs and clusters with the insect CRY2 subfamily. EsCRY has the canonical bipartite CRY structure, with a conserved N-terminal domain and a highly divergent C-terminus, that bears several binding motifs, some of them shared with insect CRY2 and others peculiar for EsCRY. We have evaluated the temporal expression of Escry both at mRNA and protein levels in individuals harvested from the Ross Sea at different times throughout the 24 h cycle during the Antarctic summer. We observed a daily fluctuation in abundance for Escry mRNA in the head, with high levels around 06:00 h, which is not mirrored by a cycle in the corresponding protein. Our findings represent a first step toward establishing the presence of an endogenous circadian time-keeping mechanism that might allow this organism to synchronize its physiology and behavior to the Antarctic light regimes. (Author correspondence: bru@unife.it or rodolfo.costa@unipd.it)

Temporal patterns of lipoperoxidation and antioxidant enzymes are modified in the hippocampus of vitamin A‐deficient rats

Animals can adapt their behavior to predictable temporal fluctuations in the environment through both, memory-and-learning processes and an endogenous time-keeping mechanism. Hippocampus plays a key role in memory and learning and is especially susceptible to oxidative stress. In compensation, antioxidant enzymes activity, such as Catalase (CAT) and Glutathione peroxidase (GPx), has been detected in this brain region. Daily rhythms of antioxidant enzymes activity, as well as of glutathione and lipid peroxides levels, have been described in brain. Here, we investigate day/night variations in lipoperoxidation, CAT, and GPx expression and activity, as well as the temporal fluctuations of two key components of the endogenous clock, BMAL1 and PER1, in the rat hippocampus and evaluate to which extent vitamin A deficiency may affect their amplitude or phase. Holtzman male rats from control, vitamin A-deficient, and vitamin A-refed groups were sacrificed throughout a 24-h period. Daily levels of clock proteins, lipoperoxidation, CAT and GPx mRNA, protein, and activity, were determined in the rat hippocampus obtained every 4 or 5 h. Gene expression of RARalpha and RXRbeta was also quantified in the hippocampus of the three groups of rats. Our results show significant daily variations of BMAL1 and PER1 protein expression. Rhythmic lipoperoxidation, CAT, and GPx, expression and activity, were also observed in the rat hippocampus. Vitamin A deficiency reduced RXRbeta mRNA level, as well as the amplitude of BMAL1 and PER1 daily oscillation, phase-shifted the daily peak of lipoperoxidation, and had a differential effect on the oscillating CAT and GPx mRNA, protein, and activity. Learning how vitamin A deficiency affects the circadian gene expression in the hippocampus may have an impact on the neurobiology, nutritional and chronobiology fields, emphasizing for the first time the importance of nutritional factors, such as dietary micronutrients, in the regulation of circadian parameters in this brain memory-and-learning-related region.

Circadian Genes Are Expressed during Early Development in Xenopus laevis

BackgroundCircadian oscillators are endogenous time-keeping mechanisms that drive twenty four hour rhythmic changes in gene expression, metabolism, hormone levels, and physical activity. We have examined the developmental expression of genes known to regulate circadian rhythms in order to better understand the ontogeny of the circadian clock in a vertebrate.Methodology/Principal FindingsIn this study, genes known to function together in part of the core circadian oscillator mechanism (xPeriod1, xPeriod2, and xBmal1) as well as a rhythmic, clock-controlled gene (xNocturnin) were analyzed using in situ hybridization in embryos from neurula to late tailbud stages. Each transcript was present in the developing nervous system in the brain, eye, olfactory pit, otic vesicle and at lower levels in the spinal cord. These genes were also expressed in the developing somites and heart, but at different developmental times in peripheral tissues (pronephros, cement gland, and posterior mesoderm). No difference was observed in transcript levels or localization when similarly staged embryos maintained in cyclic light were compared at two times of day (dawn and dusk) by in situ hybridization. Quantitation of xBmal1 expression in embryonic eyes was also performed using qRT-PCR. Eyes were isolated at dawn, midday, dusk, and midnight (cylic light). No difference in expression level between time-points was found in stage 31 eyes (p = 0.176) but stage 40 eyes showed significantly increased levels of xBmal1 expression at midnight (RQ = 1.98+/−0.094) when compared to dawn (RQ = 1+/−0.133; p = 0.0004).Conclusions/SignificanceWe hypothesize that when circadian genes are not co-expressed in the same tissue during development that it may indicate pleiotropic functions of these genes that are separate from the timing of circadian rhythm. Our results show that all circadian genes analyzed thus far are present during early brain and eye development, but rhythmic gene expression in the eye is not observed until after stage 31 of development.

The curse of physiology—challenges and opportunities in the interpretation of geochemical data from mollusk shells

Physiology corrupts geochemical records of mollusk shells in many ways, e.g., by actively controlling the incorporation of trace elements in the skeleton. However, the effects of variable biomineralization rates and growth cessation have largely remained unconsidered. Mediated by endogenous timekeeping mechanisms, mollusks stop growing skeletal material on a regular basis ranging from ultradian to annual timescales. During growth cessation, the shells do not record environmental conditions. Shell growth also stops when environmental conditions are beyond the physiological tolerance of the organism, e.g., above and below genetically determined, species-specific thermal extremes where shell growth slows and eventually ceases. Such growth disruptions can occur at non-periodic time intervals. Due to growth retardations and halts, proxy records of mollusk shells are thus incomplete, and reconstructed environmental amplitudes prone to truncation. Furthermore, environmental records are biased toward the physiological optimum of the animal. Favorable environmental conditions increase shell growth, whereas adverse environmental conditions result in reduced shell production and lowered overall metabolism. Not least, the duration of the growing season and overall growth rate decrease as the mollusk grows older. Mathematical modeling approaches can significantly improve proxy records obtained from mollusk shells. For example, if the duration of growth cessation is known, it may be possible to model the missing environmental record. It is also fairly easy to account for age-related growth trends, or variable time-averaging in different portions of the shell. However, a major premise for a reliable interpretation of proxy records from a mollusk shell or other organisms secreting biogenic hard parts is a proper understanding of the physiology, and of course, a high-resolution record of the many different environmental factors that may influence physiology and shell growth. The present paper reviews examples from the literature, and unpublished data on how physiology influences geochemical proxy records from mollusk shells, and presents methods how to eliminate such adverse effects.

In silico analysis of the endogenous time-keeping mechanism in citrus

The endogenous time-keeping mechanism is responsible for organizing plant physiology and metabolism according to periodic environmental changes, such as diurnal cycles of light and dark and seasonal progression throughout the year. In plants, circadian rhythms control gene expression, stomatal opening, and the timing component of the photoperiodic responses, leading to enhanced fitness due to increased photosynthetic rates and biomass production. We have investigated the citrus genome databases of expressed sequence tags (EST) in order to identify genes coding for functionally characterized proteins involved in the endogenous time-keeping mechanism in Arabidopsis thaliana. Approximately 180,000 EST sequences from 53 libraries were investigated and 81 orthologs of clock components were identified. We found that the vast majority of Arabidopsis circadian clock genes are present in citrus species, although some important components are absent such as SRR1 and PRR5. Based on the identified transcripts, a model for the endogenous oscillatory mechanism of citrus is proposed. These results demonstrate the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.