Diurnal Variation In Expression Research Articles

An emerging concept of diurnal variation in expression of UDP-Glucose Pyrophosphorylase gene, (StUGPase2), in potato (Solanum tuberosum L.)

Diurnal regulation of gene expression encoding the dominant isoform of UDP-Glucose Pyrophosphorylase, StUGPase2, was studied in potato leaves at different developmental stages (pre-tuberization, tuber initiation, and tuber bulking). Across the developmental stages, the StUGPase2 steady-state mRNA level increased gradually from morning (6 AM) to noon (12 PM), followed by a transient drop at 2 PM. The mRNA level increased thereafter, and the highest transcript abundance was recorded at 8:00 PM. A sharp decline in StUGPase2 transcript abundance at 2 PM in all the stages studied is believed to be associated with the previous reports of diurnal behaviour of sucrose concentration and light responsiveness of sucrose synthase enzyme. Moreover, in all three stages, a steady state increase of StUGPase2 transcript despite a consistent decrease in irradiance after 12 PM, suggests the presence of more complex transcriptional regulation. Comparatively higher StUGPase2 transcript abundance at the tuberization initiation stage as compared to the pre and post-tuberization stages indicates the prominent role of the gene during tuber development. Bioinformatics analysis of the StUGPase2 promoter region predicted the presence of various light-responsive cis-regulatory nucleotide sequences, which are considered to be associated with the light-mediated diurnal change in StUGPase2 gene expression. This research will aid in understanding the pattern of UGPase gene regulation, which is ultimately connected to potato tuber yield and bulking attributes.

Diurnal variations in expression of photosynthesis-related proteins in the harmful Raphidophyceae Chattonella marina var. antiqua

Many biological features of Chattonella species display diurnal oscillations, but little is known about the corresponding cellular responses at the proteome level. In the present study, we hypothesized that the abundance of certain proteins would periodically fluctuate to suit the diurnal changes of biological processes in Chattonella cells. To test this hypothesis, we undertook proteomic analysis over 7 time points covering a 24-h period in C. marina var. antiqua cells at early stationary phase (12:00, 17:00, 20:00, 24:00 on day 8, and 05:00, 08:00, 12:00 on the following day). The protein profiles of C. marina var. antiqua cells collected from the diurnal cycle shared a majority of common protein spots. However, the expression of 16 out of 124 detected spots exhibited significant differences between light and dark periods and/or over the 7 time points of the diurnal cycle. The proteins of OEE, Cyt c553, and AtpB showed significantly higher expression levels in the light period, and generally were maximally expressed during the period of 12:00–17:00. The proteins of GAPDH and RPL12 generally showed higher expression levels in the dark period, and were maximally expressed during the period of 20:00–24:00. These findings support our hypothesis, and suggest that the periodic expressions of these proteins may be critical either for optimal cell utilization of resources or to maintain proper cell function throughout diurnal cycles.

Effects of feeding time on daily rhythms of neuropeptide and clock gene expression in the rat hypothalamus

Shiftworkers are exposed to several adverse health conditions, one being eating at night. Food consumption at an unnatural time-of-day is thought to be one of the main factors responsible for the increased risk of developing metabolic diseases, such as obesity and diabetes mellitus. The underlying mechanism is considered to include disruption of the circadian organization of physiology, leading to disruption of metabolism. When food is consumed at night, the hypothalamus, a brain region central to homeostasis, receives contradicting input from the central clock and the systemic circulation. This study investigated how timing of feeding affects hypothalamic function by studying, in different hypothalamic nuclei, expression of clock genes and key neuropeptide genes involved in energy metabolism, including orexin, melanin-concentrating hormone (MCH) and neuropeptide Y. Animals with food available ad libitum showed diurnal variation in the expression of clock genes Per1 and Per2 in the perifornical area and arcuate nucleus. Clock gene rhythms were lost in both nuclei when food was restricted to the light (i.e., sleep) period. Neuropeptide genes did not display significant daily variation in either feeding groups, except for orexin-receptor 1 in ad libitum animals. Analysis of genes involved in glutamatergic and GABAergic signaling did not reveal diurnal variation in expression, nor effects of feeding time. In conclusion, feeding at the ‘wrong’ time-of-day not only induces desynchronization between brain and body clocks but also within the hypothalamus, which may contribute further to the underlying pathology of metabolic dysregulation.

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni)

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.

Diurnal variation in probability of death following self-poisoning in Sri Lanka--evidence for chronotoxicity in humans

The absorption, distribution, metabolism and elimination of medicines are partly controlled by transporters and enzymes with diurnal variation in expression. Dose timing may be important for maximizing therapeutic and minimizing adverse effects. However, outcome data for such an effect in humans are sparse, and chronotherapeutics is consequently less practised. We examined a large prospective Sri Lankan cohort of patients with acute poisoning to seek evidence of diurnal variation in the probability of survival. In all, 14 840 patients admitted to hospital after yellow oleander (Cascabela thevetia) seed or pesticide [organophosphorus (OP), carbamate, paraquat, glyphosate] self-poisoning were investigated for variation in survival according to time of ingestion. We found strong evidence that the outcome of oleander poisoning was associated with time of ingestion (P < 0.001). There was weaker evidence for OP insecticides (P = 0.041) and no evidence of diurnal variation in the outcome for carbamate, glyphosate and paraquat pesticides. Compared with ingestion in the late morning, and with confounding by age, sex, time of and delay to hospital presentation and year of admission controlled, case fatality of oleander poisoning was over 50% lower following evening ingestion (risk ratio = 0.40, 95% confidence interval 0.26-0.62). Variation in dose across the day was not responsible. We have shown for the first time that timing of poison ingestion affects survival in humans. This evidence for chronotoxicity suggests chronotherapeutics should be given greater attention in drug development and clinical practice.

Role of Vagal Innervation in Diurnal Rhythm of Intestinal Peptide Transporter 1 (PEPT1)

BackgroundProtein is absorbed predominantly as di/tripeptides via H+/peptide cotransporter-1 (PEPT1). We demonstrated previously diurnal variations in expression and function of duodenal and jejunal but not ileal PEPT1; neural regulation of this pattern is unexplored. HypothesisComplete abdominal vagotomy abolishes diurnal variations in gene expression and transport function of PEPT1. MethodsTwenty-four rats maintained in a 12-h light/dark room [6AM–6PM] underwent abdominal vagotomy; 24 other rats were controls. Four weeks later, mucosal levels of mRNA and protein were measured at 9AM, 3PM, 9PM, and 3AM (n = 6 each) by quantitative real-time PCR and Western blots, respectively; transporter-mediated uptake of dipeptide (Gly–Sar) was measured by the everted-sleeve technique. ResultsDiurnal variation in mRNA, as in controls, was retained post-vagotomy in duodenum and jejunum (peak at 3PM, p < 0.05) but not in ileum. Diurnal variations in expression of protein and Gly–Sar uptake, however, were absent post-vagotomy (p > 0.3). Similar to controls, maximal uptake was in jejunum after vagotomy (Vmax, nmol/cm/min: jejunum vs. duodenum and ileum; 163 vs. 88 and 71 at 3AM; p < 0.04); Km remained unchanged. ConclusionsVagal innervation appears to mediate in part diurnal variations in protein expression and transport function of PEPT1, but not diurnal variation in mRNA expression of PEPT1.

Abstract 5383: The Circadian Clock within the Cardiomyocyte is a Novel Mechanism Regulating Myocardial Triglyceride Metabolism

Virtually every mammalian cell, including the cardiomyocyte, has an intrinsic molecular circadian clock. The cardiomyocyte circadian clock has recently been shown to directly mediate diurnal variations in myocardial gene expression, metabolism, and contractile function. This includes modulation of the heart’s acute transcriptional response to increased circulating fatty acids, in a time-of-day-dependent manner. We therefore hypothesized that disruption of the cardiomyocyte circadian clock would impair metabolic adaptation of the heart to high fat feeding. Wild type (WT) and cardiomyocyte-specific circadian clock mutant (CCM) mice were fed either a control or high fat diet (10% and 45% calories from fat, respectively) for 16 weeks, after which myocardial adaptation was assessed at transcriptional, metabolic, and functional levels. Ex vivo mouse heart perfusions in the working mode revealed that high fat feeding induced alterations in myocardial metabolism ( e.g. reduced carbohydrate oxidation; p&lt;0.01) and contractile function ( e.g. depressed cardiac power; p&lt;0.01) in WT, but not CCM, hearts. Furthermore, the ability to decrease reliance on endogenous substrate utilization during an acute increase in fatty acid availability ex vivo was abolished in hearts from CCM mice fed the high fat diet (p&lt;0.05), suggesting potential alterations in triglyceride metabolism. Consistent with the latter, microarray analysis revealed that the cardiomyocyte circadian clock regulates expression of several genes involved in triglyceride turnover. For example, diurnal variations in expression of diacylglycerol acyltransferase 2, which promotes lipogenesis, and rates of myocardial triglyceride synthesis both peak in the middle of the active phase (2- and 3- fold, trough-to-peak, respectively; p&lt;0.05), which are abolished in CCM hearts. These data provide direct evidence that the cardiomyocyte circadian clock regulates myocardial triglyceride metabolism at multiple levels. We postulate that disruption of this intramyocellular mechanism may contribute to altered myocardial metabolism and contractile dysfunction observed during cardiac hypertrophy, diabetes mellitus, and/or shift work.

Coordinated diurnal regulation of genes from the Dlk1–Dio3 imprinted domain: implications for regulation of clusters of non-paralogous genes

The functioning of the genome is tightly related to its architecture. Therefore, understanding the relationship between different regulatory mechanisms and the organization of chromosomal domains is essential for understanding genome regulation. The majority of imprinted genes are assembled into clusters, share common regulatory elements, and, hence, represent an attractive model for studies of regulation of clusters of non-paralogous genes. Here, we investigated the relationship between genomic imprinting and diurnal regulation of genes from the imprinted domain of mouse chromosome 12. We compared gene expression patterns in C57BL/6 mice and congenic mice that carry the imprinted region from a Mus musculus molossinus strain MOLF/Ei. In the C57BL/6 mice, a putative enhancer/oscillator regulated the expression of only Mico1/Mico1os, whereas in the congenic mice its influence was spread onto Rtl1as, Dio3 and Dio3os, i.e. the distal part of the imprinted domain, resulting in coordinated diurnal variation in expression of five genes. Using additional congenic strains we determined that in C57BL/6 the effect of the putative enhancer/oscillator was attenuated by a linked dominant trans-acting factor located in the distal portion of chromosome 12. Our data demonstrate that (i) in adult organs, mRNA levels of several imprinted genes vary during the day, (ii) genetic variation may remove constraints on the influence of an enhancer and lead to spreading of its effect onto neighboring genes, thereby generating genotype-dependent expression patterns and (iii) different regulatory mechanisms within the same domain act independently and do not seem to interfere with each other.

Circadian Rhythms of Isoprene Biosynthesis in Grey Poplar Leaves

Isoprene (2-methyl-1,3-butadiene) emission varies diurnally in different species. In poplar (Populus spp.), it has recently been shown that the gene encoding the synthesizing enzyme for isoprene, isoprene synthase (ISPS), displays diurnal variation in expression. Working on shoot cultures of Grey poplar (Populus x canescens) placed under a different light regime in phytochambers, we showed that these variations in PcISPS gene expression, measured by quantitative real-time polymerase chain reaction, are not only due to day-night changes, but also are linked to an internal circadian clock. Measurement of additional selected isoprenoid genes revealed that phytoene synthase (carotenoid pathway) displays similar fluctuations, whereas 1-deoxy-d-xylulose 5-phosphate reductoisomerase, possibly the first committed enzyme of the 1-deoxy-d-xylulose 5-phosphate pathway, only shows light regulation. On the protein level, it appeared that PcISPS activity and protein content became reduced under constant darkness, whereas under constant light, activity and protein content of this enzyme were kept high. In contrast, isoprene emission rates under continuous irradiation displayed circadian changes as is the case for gene expression of PcISPS. Furthermore, binding assays with Arabidopsis (Arabidopsis thaliana) late elongated hypocotyl, a transcription factor of Arabidopsis involved in circadian regulation, clearly revealed the presence of circadian-determining regulatory elements in the promoter region of PcISPS.

Seasonal and diurnal variations in gene expression in the desert legume Retama raetam

AbstractStudying plants growing naturally within their habitat can contribute greatly to our understanding of molecular and biochemical processes involved in the response of plants to changes in environmental conditions. Seasonal and diurnal variations in the expression of several transcripts involved in the defence of plants against abiotic stresses, i.e. transcripts encoding dehydrins, heat shock proteins, and antioxidative enzymes, were followed in Retama raetam plants that grow naturally within an arid dune ecosystem. These were associated with changes in environmental parameters simultaneously recorded at the research sites. It was found that the expression pattern of some transcripts correlated with changes in environmental conditions in the different plants during the year. In contrast, the expression pattern of other transcripts appeared to be plant specific and may be associated with phenotypic variability (plasticity). Transcripts encoding different heat shock proteins were induced in a co‐ordinated manner, suggesting that their corresponding products function as a chaperone network. Measurements of photosynthetic activity revealed a diurnal cycle in R. raetam. Photosynthesis was highest during early morning, declined toward the stressful midday hours and recovered at late afternoon. The present analysis suggests that R. raetam uses a combination of different avoidance strategies in co‐ordination with active defence mechanisms to withstand the stressful conditions that prevail within the desert ecosystem.

Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots.

Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. 15N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants. To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake-deficient yeast mutant. Uptake studies with 14C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between </=0.5 and 40 microM. Correlation of gene expression with 15NH4+ uptake into plant roots showed that nitrogen supply and time of day differentially regulated the individual carriers. Transcript levels of AtAMT1;1, which possesses an affinity in the nanomolar range, steeply increased with ammonium uptake in roots when nitrogen nutrition became limiting, whereas those of AtAMT1;3 increased slightly, with AtAMT1;2 being more constitutively expressed. All three ammonium transporters showed diurnal variation in expression, but AtAMT1;3 transcript levels peaked with ammonium uptake at the end of the light period, suggesting that AtAMT1;3 provides a link between nitrogen assimilation and carbon provision in roots. Our results show that high-affinity ammonium uptake in roots is regulated in relation to the physiological status of the plant at the transcriptional level and by substrate affinities of individual members of the AMT1 gene family.

Temporal and photoregulated expression of five tomato phytochrome genes.

Quantitative measurements of the absolute amounts of mRNAs transcribed from each of five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE, PHYF) throughout the life cycle of a tomato plant and in response to changes in ambient light conditions are reported here. From their lowest level in unimbibed seed, all five transcripts increase by from 10- to 1000-fold during the first 24 h following the onset of imbibition, both in continuous darkness and in a greenhouse. In a greenhouse and on a whole-plant basis, all continue to increase throughout day 6, after which all but PHYE decline over the next week to a plateau at about one-half of the maximal value. PHYE mRNA differs in that in continues to increase in abundance during the first 2-3 weeks and thereafter remains at that maximal level. In adult plants, on a whole-plant basis and in decreasing order of abundance, PHYA, PHYB1, PHYE, PHYB2, and PHYF transcripts were present at approximately 120, 40, 40, 15, and 8 mumol microgram-1 of poly(A)(+)-enriched RNA, respectively. The data are consistent with the demonstrated roles of phytochromes A and B1 during seedling development and lead to the suggestion that phytochrome E might have a more important role in mature plants. Somewhat unexpectedly, PHYA and PHYB2 expression patterns are very similar. In seedlings, PHYA and PHYB2 exhibit the greatest increase in expression following a light-to-dark transition, as well as the greatest decrease following a dark-to-light transition. PHYA and PHYB2 are also similar in that both exhibit comparable variation on a natural diurnal cycle, while PHYB1 also exhibits variation but with a markedly different phase. The diurnal variation in expression of PHYA, PHYB1 and PHYB2 is consistent with the possibility that one or more of the phytochromes they encode is important with respect not only to photoperiodic behavior but also to the regulation of other events whose photosensitivity varies during a diurnal cycle.

Glucocorticoid regulation of neuropeptide mRNAs in the rat striatum

Expression of the preprotachykinin (PPT) mRNA and of the preproenkephalin (PPE) mRNA in the rat striatum has been assessed by in situ hybridization. The results demonstrate that the PPT mRNA is regulated by glucocorticoids such that adrenalectomized (ADX) animals replaced with corticosterone for 5 days expressed higher levels of the mRNA than ADX animals. The corticosterone-induced increase in striatal PPT mRNA was evident after 16 h, but not after 2 h, of corticosterone treatment of ADX animals. Elevation of circulating corticosterone levels in intact rats by acute restraint stress, or by corticosterone injection did not change the level of PPT mRNA in the striatum. In intact rats there was a diurnal variation in the level of striatal PPE mRNA expression; adrenalectomy resulted in a decrease in the mRNA level and did not abolish the diurnal variation in expression. The level of PPT mRNA in the striatum was also decreased in response to ADX, but there were no significant diurnal changes in the expression of the PPT mRNA either in the intact or in the ADX animals.

Glucocorticoid Regulation of Preproenkephalin Messenger Ribonucleic Acid in the Rat Striatum*

Glucocorticoids regulate the level of preproenkephalin mRNA expression in a number of cell systems. This study investigated the expression of preproenkephalin mRNA in the brain and its regulation by glucocorticoids in vivo. Two different methods for mRNA quantitation were employed. Total RNA isolated from dissected brain tissue was analyzed in an RNAse T2 protection assay. In addition, we have used in situ hybridization to brain sections to assess the expression of preproenkephalin mRNA. The results demonstrate that in the striatum the preproenkephalin mRNA is expressed at a high level and is regulated by glucocorticoids. There is a decrease in striatal preproenkephalin mRNA after adrenalectomy (ADX), and ADX animals replaced with corticosterone express higher levels of striatal preproenkephalin mRNA than ADX animals. By in situ hybridization we have determined that the corticosterone-induced increase in striatal preproenkephalin mRNA is evident after 16 h, but not after 2 h, of corticosterone replacement of ADX animals. Elevation of circulating corticosterone levels in intact rats by 2-3 h of restraint stress, a multiple daily stress regimen over 1-21 days, or corticosterone injection did not change the level of preproenkephalin mRNA in the striatum; however, a single 16-h restraint stress resulted in a decreased level of expression. In intact rats there was a diurnal variation in the level of striatal preproenkephalin mRNA expression; ADX resulted in a decrease in the mRNA level, but did not abolish the diurnal variation in expression. Thus, while there is clearly regulation of striatal preproenkephalin mRNA by glucocorticoids, our results provide evidence for regulation by nonglucocorticoid mechanisms as well.