Clock Regulation Research Articles

Photoperiodic and clock regulation of the vitamin A pathway in the brain mediates seasonal responsiveness in the monarch butterfly

Seasonal adaptation to changes in light:dark regimes (i.e., photoperiod) allows organisms living at temperate latitudes to anticipate environmental changes. In nearly all animals studied so far, the circadian system has been implicated in measurement and response to the photoperiod. In insects, genetic evidence further supports the involvement of several clock genes in photoperiodic responses. Yet, the key molecular pathways linking clock genes or the circadian clock to insect photoperiodic responses remain largely unknown. Here, we show that inactivating the clock in the North American monarch butterfly using loss-of-function mutants for the circadian activators CLOCK and BMAL1 and the circadian repressor CRYPTOCHROME 2 abolishes photoperiodic responses in reproductive output. Transcriptomic approaches in the brain of monarchs raised in long and short photoperiods, summer monarchs, and fall migrants revealed a molecular signature of seasonal-specific rhythmic gene expression that included several genes belonging to the vitamin A pathway. We found that the rhythmic expression of these genes was abolished in clock-deficient mutants, suggesting that the vitamin A pathway operates downstream of the circadian clock. Importantly, we showed that a CRISPR/Cas9-mediated loss-of-function mutation in the gene encoding the pathway's rate-limiting enzyme, ninaB1, abolished photoperiod responsiveness independently of visual function in the compound eye and without affecting circadian rhythms. Together, these results provide genetic evidence that the clock-controlled vitamin A pathway mediates photoperiod responsiveness in an insect. Given previously reported seasonal changes associated with this pathway in the mammalian brain, our findings suggest an evolutionarily conserved function of vitamin A in animal photoperiodism.

Circadian clock regulates the shape and content of dendritic spines in mouse barrel cortex.

Circadian rhythmicity affects neuronal activity induced changes in the density of synaptic contacts and dendritic spines, the most common location of synapses, in mouse somatosensory cortex. In the present study we analyzed morphology of single- and double-synapse spines under light/dark (12:12) and constant darkness conditions. Using serial electron micrographs we examined the shape of spines (stubby, thin, mushroom) and their content (smooth endoplasmic reticulum, spine apparatus), because these features are related to the maturation and stabilization of spines. We observed significant diurnal and circadian changes in the shape of spines that are differentially regulated: single-synapse spines remain under circadian clock regulation, while changes of double-synapse spines are driven by light. The thin and mushroom single-synapse spines, regardless of their content, are more stable comparing with the stubby single-synapse spines that show the greatest diversity. All types of double-synapse spines demonstrate a similar level of stability. In light/dark regime, formation of new mushroom single-synapse spines occurs, while under constant darkness new stubby single-synapse spines are formed. There are no shape preferences for new double-synapse spines. Diurnal and circadian alterations also concern spine content: both light exposure and the clock influence translocation of smooth endoplasmic reticulum from dendritic shaft to the spine. The increasing number of mushroom single-synapse spines and the presence of only those mushroom double-synapse spines that contain spine apparatus in the light phase indicates that the exposure to light, a stress factor for nocturnal animals, promotes enlargement and maturation of spines to increase synaptic strength and to enhance the effectiveness of neurotransmission.

Hepatic lipid metabolism is affected by a daily 3-meal pattern with varying dietary crude protein with a pig model.

The present study was conducted to evaluate the effects of 3 meals administered daily with varying dietary crude protein (CP) contents on hepatic lipid metabolism with a pig model. Pigs were divided into 3 groups according to the following feeding patterns: feeding a basal CP diet 3 times daily (3C); feeding a high CP diet for breakfast, the basal CP diet for lunch, and a low CP diet for dinner (HCL); and feeding the low CP diet for breakfast, the basal CP diet for lunch, and the high protein diet for dinner (LCH). Three groups took equivalent diet per meal ensuring that every pig was fed with similar dietary formulae daily. Results showed that HCL feeding pattern reduced the relative kidney weight (P < 0.05), and LCH feeding pattern increased the relative liver weight of pigs (P < 0.05) when compared with those in the 3C group. Plasma urea nitrogen (P < 0.01) and lipase (P < 0.05) decreased in the HCL group but increased in the LCH group. Both HCL and LCH feeding patterns reduced plasma triglycerides (P < 0.01), non-esterified fatty acids (NEFA) (P < 0.01), and hepatic crude fat (0.05 < P < 0.10) of pigs. Real-time quantitative PCR (RT-qPCR) results showed that dynamic feeding patterns down-regulated (P < 0.05) the mRNA level of lipid metabolism related genes, including adipose triglyceride lipase (ATGL), acetyl-CoA carboxylase (ACCα), liver X receptor (LXRα) in the liver, and negatively regulate elements of circadian clock, including period 1 (Per1), period 2 (Per2), cryptochrome (Cry2), which in turn, upregulated (P < 0.05) the protein expression of positive regulate element brain and muscle Arnt-like 1 (BMAL1) when compared with 3C group. Overall, our findings suggested that dynamic feeding patterns may affect hepatic lipid metabolism via regulation of the circadian clock.

A Critical Appraisal of Strategies to Optimize Vitamin D Status in Germany, a Population with a Western Diet.

During the last decade, our scientific knowledge of the pleiotropic biological effects of vitamin D metabolites and their relevance to human health has expanded widely. Beyond the well-known key role of vitamin D in calcium homeostasis and bone health, it has been shown that vitamin D deficiency is associated with a broad variety of independent diseases, including several types of cancer, and with increased overall mortality. Moreover, recent findings have demonstrated biological effects of the vitamin D endocrine system that are not mediated via activation of the classical nuclear vitamin D receptor (VDR) by binding with high affinity to its corresponding ligand, the biologically active vitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D). In contrast, many of these new biological effects of vitamin D compounds, including regulation of the circadian clock and many metabolic functions, are mediated by other vitamin D metabolites, including 20-hydroxyvitamin D and 20,23-dihydroxyvitamin D, and involve their binding to the aryl hydrocarbon receptor (AhR) and retinoid-orphan receptor (ROR). In most populations, including the German population, UVB-induced cutaneous vitamin D production is the main source for fulfilling the human body’s requirements of vitamin D. However, this causes a dilemma because solar or artificial UVR exposure is associated with skin cancer risk. In addition to UVB-induced vitamin D production in skin, in humans, there are two other possible sources of vitamin D: from diet and supplements. However, only a few natural foods contain substantial amounts of vitamin D, and in most populations, the dietary source of vitamin D cannot fulfill the body´s requirements. Because an increasing body of evidence has convincingly demonstrated that vitamin D deficiency is very common worldwide, it is the aim of this paper to (i) give an update of the vitamin D status in a population with a western diet, namely, the German population, and to (ii) develop strategies to optimize the vitamin D supply that consider both the advantages as well as the disadvantages/risks of different approaches, including increasing vitamin D status by dietary intake, by supplements, or by UVB-induced cutaneous synthesis of vitamin D.

Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock.

Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.

Regulation of the Neurospora Circadian Clock by the Spliceosome Component PRP5.

Increasing evidence has pointed to the connection between pre-mRNA splicing and the circadian clock; however, the underlying mechanisms of this connection remain largely elusive. In the filamentous fungus Neurospora crassa, the core circadian clock elements comprise White Collar 1 (WC-1), WC-2 and FREQUENCY (FRQ), which form a negative feedback loop to control the circadian rhythms of gene expression and physiological processes. Previously, we have shown that in Neurospora, the pre-mRNA splicing factors Pre-mRNA-processing ATP-dependent RNA helicase 5 (PRP5), protein arginine methyl transferase 5 (PRMT5) and snRNA gene U4-2 are involved in the regulation of splicing of frq transcripts, which encode the negative component of the circadian clock system. In this work we further demonstrated that repression of spliceosomal component sRNA genes, U5, U4-1, and prp5, affected the circadian conidiation rhythms. In a prp5 knockdown strain, the molecular rhythmicity was dampened. The expression of a set of snRNP genes including prp5 was up-regulated in a mutant strain lacking the clock component wc-2, suggesting that the function of spliceosome might be under the circadian control. Among these snRNP genes, the levels of prp5 RNA and PRP5 protein oscillated. The distribution of PRP5 in cytosol was rhythmic, suggesting a dynamic assembly of PRP5 in the spliceosome complex in a circadian fashion. Silencing of prp5 caused changes in the transcription and splicing of NCU09649, a clock-controlled gene. Moreover, in the clock mutant frq9, the rhythmicity of frq I-6 splicing was abolished. These data shed new lights on the regulation of circadian clock by the pre-RNA splicing, and PRP5 may link the circadian clock and pre-RNA splicing events through mediating the assembly and function of the spliceosome complex.

An All-Digital Fused PLL-Buck Architecture for 82% Average V dd-Margin Reduction in a 0.6-to-1.0-V Cortex-M0 Processor

Traditional digital systems employ independent loops to control supply voltage ( ${\mathit {V}}_{\text {dd}}$ ) and clock frequency ( $f_{\text {clk}}$ ). A clock regulation loop, for instance, a phase-locked loop (PLL), locks the system clock to a reference clock (REFCLK). Concurrently, a voltage regulation loop sets ${\mathit {V}}_{\text {dd}}$ to a target value under rapidly varying load current or line-side disturbances. Limitations in voltage regulator loop bandwidth result in significant ${\mathit {V}}_{\text {dd}}$ fluctuation, leading to timing slack degradation in digital voltage domains. Maintaining timing slack in the presence of supply noise or temperature (T) variation incurs energy-wasteful ${\mathit {V}}_{\text {dd}}$ guardbands. In this article, we present a unified clock and power (UniCaP) architecture that instantaneously adapts $f_{\text {clk}}$ to ${\mathit {V}}_{\text {dd}}$ and $T$ variations, requiring minimal guardbands. UniCaP absorbs ${\mathit {V}}_{\text {dd}}$ control into the clock regulation loop, using it to adjust $f_{\text {clk}}$ and lock system clock to REFCLK. UniCaP enables all-digital construction with aggressive ${\mathit {V}}_{\text {dd}}$ guardband reduction and the capability for on-the-fly dynamic voltage and frequency scaling (DVFS) events. We deployed the UniCaP architecture on a 65-nm buck converter test chip powering a 0.6–1.0-V Cortex-M0 microprocessor with autonomous transition between continuous and discontinuous conduction modes (CCM/DCM) in order to support a wide load current range. The UniCaP test chip demonstrates 82% average ${\mathit {V}}_{\text {dd}}$ guardband reduction, without any performance loss from adaptive clocking, and temperature margin reductions of 40–55 mV.

Transcriptomic Analysis of Differentially Expressed Genes and Alternative Splicing Events Associated with Crassulacean Acid Metabolism in Orchids

Phalaenopsis equestris is an obligate crassulacean acid metabolism (CAM) plant with high ornamental and economic value. CAM photosynthesis is associated with drought tolerance and efficient water utilization, which enhances the survival rate of CAM plants in arid environments. The identification and analysis of CAM-related genes will be helpful to improve our understanding of the regulatory mechanisms of CAM metabolism. In this study, we analyzed RNA-Seq data to identify differentially expressed genes (DEGs) between circadian day and night in P. equestris leaves then performed GO and KEGG functional enrichment analysis. The pathways that were significantly enriched among these DEGs included carbon fixation, circadian clock regulation, glucose metabolism, photosynthesis, and plant hormone signaling. We also used PacBio long-read Iso-Seq technology, which identified many alternative splicing events for key genes in CAM-related pathways, including carbon fixation, circadian clock regulation, and stomatal movement. These findings suggested that alternative splicing events might be involved in CAM metabolism. Many unknown or uncharacterized genes were also found to be potentially involved in CAM metabolism. For example, the Peq000162 gene encodes a protein belonging to the LdpA (light-dependent period) iron-sulfur protein family, and it was found to generate many alternatively spliced products. These findings shed light on CAM metabolic mechanisms in P. equestris along with the molecular functions of key CAM genes. Ultimately, the information may help enhance crop yield and drought tolerance through the introduction of CAM features into C3 crops.

TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation.

The Arabidopsis genome contains three genes encoding proteins of the TRANSPARENT TESTA GLABRA 1 (TTG1) WD-repeat (WDR) subfamily. TTG1 is a known regulator of epidermal cell differentiation and pigment production, while LIGHT-REGULATED WD1 and LIGHT-REGULATED WD2 are known regulators of the circadian clock. Here, we discovered a new central role for TTG1 WDR proteins as regulators of the circadian system, as evidenced by the lack of detectable circadian rhythms in a triple lwd1 lwd2 ttg1 mutant. This shows that there has been subfunctionalization via protein changes within the angiosperms, with some TTG1 WDR proteins developing a stronger role in circadian clock regulation while losing the protein characteristics essential for pigment production and epidermal cell specification, and others weakening their ability to drive circadian clock regulation. Our work shows that even where proteins are very conserved, small changes can drive big functional differences.

Circadian Clock Regulation of Hepatic Energy Metabolism Regulatory Circuits.

The liver is a critical organ of energy metabolism. At least 10% of the liver transcriptome demonstrates rhythmic expression, implying that the circadian clock regulates large programmes of hepatic genes. Here, we review the mechanisms by which this rhythmic regulation is conferred, with a particular focus on the transcription factors whose actions combine to impart liver- and time-specificity to metabolic gene expression.

In silico Identification of a Molecular Circadian System With Novel Features in the Crustacean Model Organism Parhyale hawaiensis.

The amphipod Parhyale hawaiensis is a model organism of growing importance in the fields of evolutionary development and regeneration. A small, hardy marine crustacean that breeds year-round with a short generation time, it has simple lab culture requirements and an extensive molecular toolkit including the ability to generate targeted genetic mutant lines. Here we identify canonical core and regulatory clock genes using genomic and transcriptomic resources as a first step in establishing this species as a model in the field of chronobiology. The molecular clock of P. hawaiensis lacks orthologs of the canonical circadian genes cryptochrome 1 and timeless, in common with the mammalian system but in contrast to many arthropods including Drosophila melanogaster. Furthermore the predicted CLOCK peptide is atypical and CRY2 shows an extended 5′ region of unknown function. These results appear to be shared by two other amphipod species.

Time after time: circadian clock regulation of intestinal stem cells.

Daily fluctuations in animal physiology, known as circadian rhythms, are orchestrated by a conserved molecular timekeeper, known as the circadian clock. The circadian clock forms a transcription-translation feedback loop that has emerged as a central biological regulator of many 24-h processes. Early studies of the intestine discovered that many digestive functions have a daily rhythm and that intestinal cell production was similarly time-dependent. As genetic methods in model organisms have become available, it has become apparent that the circadian clock regulates many basic cellular functions, including growth, proliferation, and differentiation, as well as cell signalling and stem cell self-renewal. Recent connections between circadian rhythms and immune system function, and between circadian rhythms and microbiome dynamics, have also been revealed in the intestine. These processes are highly relevant in understanding intestinal stem cell biology. Here we describe the circadian clock regulation of intestinal stem cells primarily in two model organisms: Drosophila melanogaster and mice. Like all cells in the body, intestinal stem cells are subject to circadian timing, and both cell-intrinsic and cell-extrinsic circadian processes contribute to their function.

Evolution of the visual sensory system in cichlid fishes from crater lake Barombi Mbo in Cameroon.

In deep-water animals, the visual sensory system is often challenged by the dim-light environment. Here, we focus on the molecular mechanisms involved in rapid deep-water adaptations. We examined visual system evolution in a small-scale yet phenotypically and ecologically diverse adaptive radiation, the species flock of cichlid fishes in deep crater lake Barombi Mbo in Cameroon, West Africa. We show that rapid adaptations of the visual system to the novel deep-water habitat primarily occurred at the level of gene expression changes rather than through nucleotide mutations, which is compatible with the young age of the radiation. Based on retinal bulk RNA sequencing of all eleven species, we found that the opsin gene expression pattern was substantially different for the deep-water species. The nine shallow-water species feature an opsin palette dominated by the red-sensitive (LWS) opsin, whereas the two unrelated deep-water species lack expression of LWS and the violet-sensitive (SWS2B) opsin, thereby shifting the cone sensitivity to the centre of the light spectrum. Deep-water species further predominantly express the green-sensitive RH2Aα over RH2Aβ. We identified one amino acid substitution in the RH2Aα opsin specific to the deep-water species. We finally performed a comparative gene expression analysis in retinal tissue of deep- vs. shallow-water species. We thus identified 46 differentially expressed genes, many of which are associated with functions in vision, hypoxia management or circadian clock regulation, with some of them being associated with human eye diseases.

Multistability and basin stability in coupled pendulum clocks.

In this paper, we investigate the phenomenon of multistability and the concept of basin stability in two coupled pendula with escapement mechanisms, suspended on horizontally oscillating beam. The dynamics of a single pendulum clock is studied and described, showing possible responses of the unit. The basin stability maps are discussed in two-parameters plane, where we vary both the system's stiffness as well as the damping. The possible attractors for the investigated clocks are discussed, showing that different patterns of synchronization and desynchronization can occur. The oscillators may completely synchronize in one of the three possible combinations (including inphase and antiphase ones), practically synchronize with some fluctuations or stay in the irregular pattern, which includes chaotic motion. The transitions between solutions are studied, uncovering that the road from one type of dynamics into another may become very complex. Moreover, we examine the multistability property of our model using the bifurcation diagrams and the basins of attraction maps, discussing possible scenarios in which the states co-exist. The analysis of attractors' basins uncovers complicated structure of the latter ones, exhibiting that the final behavior of investigated model may be hard to determine and trace. Our results are discussed for the cases of identical and nonidentical pendula, as well as light and heavy beam, showing that depending on considered scenario, various patterns of behaviors and transitions may be observed. The research described in this paper proves that the mechanical properties of the system's suspension may play a crucial role in the possibility of the appearance of different types of attractors and that the basin stabilities of states strictly depend on the values of considered parameters.

MicroRNA-19b predicts widespread pain and posttraumatic stress symptom risk in a sex-dependent manner following trauma exposure.

Posttraumatic widespread pain (PTWP) and posttraumatic stress symptoms (PTSS) are frequent comorbid sequelae of trauma that occur at different rates in women and men. We sought to identify microRNA (miRNA) that may contribute to sex-dependent differences in vulnerability to these outcomes. Monte Carlo simulations (x10,000) identified miRNA in which predicted targeting of PTWP or PTSS genes was most enriched. Expression of the leading candidate miRNA to target PTWP/PTSS-related genes, miR-19b, has been shown to be influenced by estrogen and stress exposure. We evaluated whether peritraumatic miR-19b blood expression levels predicted PTWP and PTSS development in women and men experiencing trauma of motor vehicle collision (n = 179) and in women experiencing sexual assault trauma (n = 74). A sex-dependent relationship was observed between miR-19b expression levels and both PTWP (β = -2.41, P = 0.034) and PTSS (β = -3.01, P = 0.008) development 6 months after motor vehicle collision. The relationship between miR-19b and PTSS (but not PTWP) was validated in sexual assault survivors (β = -0.91, P = 0.013). Sex-dependent expression of miR-19b was also observed in blood and nervous tissue from 2 relevant animal models. Furthermore, in support of increasing evidence indicating a role for the circadian rhythm (CR) in PTWP and PTSS pathogenesis, miR-19b targets were enriched in CR gene transcripts. Human cohort and in vitro analyses assessing miR-19b regulation of key CR transcripts, CLOCK and RORA, supported the potential importance of miR-19b to regulating the CR pathway. Together, these results highlight the potential role that sex-dependent expression of miR-19b might play in PTWP and PTSS development after trauma/stress exposure.

Coordination of light, circadian clock with temperature: The potential mechanisms regulating chilling tolerance in rice.

Rice (Oryza sativa L.) is a major staple food crop for over half of the world's population. As a crop species originated from the subtropics, rice production is hampered by chilling stress. The genetic mechanisms of rice responses to chilling stress have attracted much attention, focusing on chilling-related gene mining and functional analyses. Plants have evolved sophisticated regulatory systems to respond to chilling stress in coordination with light signaling pathway and internal circadian clock. However, in rice, information about light-signaling pathways and circadian clock regulation and their roles in chilling tolerance remains elusive. Further investigation into the regulatory network of chilling tolerance in rice is needed, as knowledge of the interaction between temperature, light, and circadian clock dynamics is limited. Here, based on phenotypic analysis of transgenic and mutant rice lines, we delineate the relevant genes with important regulatory roles in chilling tolerance. In addition, we discuss the potential coordination mechanism among temperature, light, and circadian clock in regulating chilling response and tolerance of rice, and provide perspectives for the ongoing chilling signaling network research in rice.

CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production.

CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.

Possible application of melatonin treatment in human diseases of the biliary tract

Melatonin was discovered in 1958 by Aaron Lerner. Its name comes from the ability of melatonin to change the shape of amphibian melanophores from stellate to roundish. Starting from the 1980s, the role of melatonin in the regulation of mammalian circadian and seasonal clocks has been elucidated. Presently, several other effects have been identified in different organs. For example, the beneficial effects of melatonin in models of liver damage have been described. This review gives first a general background on experimental and clinical data on the use of melatonin in liver damage. The second part of the review focuses on the findings related to the role of melatonin in biliary functions, suggesting a possible use of melatonin therapy in human diseases of the biliary tree.

Circadian clock regulation of cone to horizontal cell synaptic transfer in the goldfish retina.

Although it is well established that the vertebrate retina contains endogenous circadian clocks that regulate retinal physiology and function during day and night, the processes that the clocks affect and the means by which the clocks control these processes remain unresolved. We previously demonstrated that a circadian clock in the goldfish retina regulates rod-cone electrical coupling so that coupling is weak during the day and robust at night. The increase in rod-cone coupling at night introduces rod signals into cones so that the light responses of both cones and cone horizontal cells, which are post-synaptic to cones, become dominated by rod input. By comparing the light responses of cones, cone horizontal cells and rod horizontal cells, which are post-synaptic to rods, under dark-adapted conditions during day and night, we determined whether the daily changes in the strength of rod-cone coupling could account entirely for rhythmic changes in the light response properties of cones and cone horizontal cells. We report that although some aspects of the day/night changes in cone and cone horizontal cell light responses, such as response threshold and spectral tuning, are consistent with modulation of rod-cone coupling, other properties cannot be solely explained by this phenomenon. Specifically, we found that at night compared to the day the time course of spectrally-isolated cone photoresponses was slower, cone-to-cone horizontal cell synaptic transfer was highly non-linear and of lower gain, and the delay in cone-to-cone horizontal cell synaptic transmission was longer. However, under bright light-adapted conditions in both day and night, cone-to-cone horizontal cell synaptic transfer was linear and of high gain, and no additional delay was observed at the cone-to-cone horizontal cell synapse. These findings suggest that in addition to controlling rod-cone coupling, retinal clocks shape the light responses of cone horizontal cells by modulating cone-to-cone horizontal cell synaptic transmission.

Photoperiod modulates growth, morphoanatomy, and linalool content in Lippia alba L. (Verbenaceae) cultured in vitro

Interactions between circadian clock regulation and metabolic responses are believed to explain the importance of rhythmic behavior in plant growth and survival. Lippia alba is an important species because of the medicinal properties of its essential oil extract. The objective of this work was to evaluate the effect of photoperiod on anatomy, growth, essential oil profile, and the expression of genes related to the synthesis of monoterpenes, sesquiterpenes, and the circadian clock in L. alba grown in vitro. The plants were cultured in vitro under different photoperiods (4, 8, 16, and 24 h of light) and irradiance of 41 μmol m−2 s−1. After 40 days of culture, results showed that L. alba presented high physiological plasticity under different photoperiods, with improved performance when exposed to continuous light. The best growth; anatomical organization of the mesophyll, stem, roots, and bundles; amount of photosynthetic pigments; photosynthetic rate; and protein synthesis occurred under a photoperiod of 24 h. The biosynthesis of linalool, the major compound, was increased under the 24-h photoperiod, possibly due to reduced geraniol synthesis. These findings allow a better understanding of how photoperiod acts in the regulation of primary and secondary metabolism, and especially with regard to the composition of essential oils. Photoperiod modulates primary metabolism, growth, morphoanatomy, photosynthesis, and essential oil content in the medicinal plant Lippia alba cultured in vitro under 4, 8, 16 or 24 h of light.