Melatonin Synthesis Pathway Research Articles

Novel melatonin-producing Bacillus safensis EH143 mitigates salt and cadmium stress in soybean.

Recently, microorganism and exogenous melatonin application has been recognized as an efficient biological tool for enhancing salt tolerance and heavy metal detoxification in agriculture crops. Thus, the goal of this study was to isolate and evaluate a novel melatonin-producing plant growth promoting bacterium. With high-throughput whole genome sequencing, phytohormone measurements, expression profiling, and biochemical analysis, we can identify a novel PGPB that produces melatonin and unravel how it promotes soybean growth and development and protects against salt and Cd stress. We identify the melatonin synthesis pathway (tryptophan→tryptamine→serotonin melatonin) of the halotolerant (NaCl > 800 mM) and heavy metal-resistant (Cd >3 mM) rhizobacterium Bacillus safensis EH143 and use it to treat soybean plants subjected to Cd and NaCl stresses. Results show that EH143 will highly bioaccumulate heavy metals and significantly improve P and Ca2+ uptake and the K+/Na+ (93%↑under salt stress) ratio while reducing Cd uptake (49% under Cd stress) in shoots. This activity was supported by the expression of the ion regulator HKT1, MYPB67, and the calcium sensors CDPK5 and CaMK1 which ultimately led to increased plant growth. EH143 significantly decreased ABA content in shoots by 13%, 20%, and 34% and increased SA biosynthesis in shoots by 14.8%, 31%, and 48.2% in control, salt, and Cd-treated plants, upregulating CYP707A1 and CYP707A2 and PAL1 and ICS, respectively. The melatonin content significantly decreased along with a reduced expression of ASMT3 following treatment with EH143; moreover, reduced expression of peroxidase (POD) and superoxide dismutase (SOD) by 134.5% and 39% under salt+Cd stress, respectively and increased level of total amino acids were observed. Whole-genome sequencing and annotation of EH143 revealed the presence of the melatonin precursor tryptophan synthase (trpA, trpB, trpS), metal and other ion regulators (Cd: cadA, potassium: KtrA and KtrB, phosphate: glpT, calcium: yloB, the sodium/glucose cotransporter: sgIT, and the magnesium transporter: mgtE), and enzyme activators (including the siderophore transport proteins yfiZ and yfhA, the SOD sodA, the catalase katA1, and the glutathione regulator KefG) that may be involved in programming the plant metabolic system. As a consequence, EH143 treatment significantly reduced the contents of lipid peroxidation (O2-, MDA, and H2O2) up to 69%, 46%, and 29% in plants under salt+Cd stress, respectively. These findings suggest that EH143 could be a potent biofertilizer to alleviate NaCl and Cd toxicity in crops and serve as an alternative substitute for exogenous melatonin application.

The Identification of Cucumber TDC Genes and Analyses of Their Expression and Functions under Abiotic Stress Conditions

Melatonin is a crucial regulator of plant growth and development as well as stress tolerance. However, we only have a limited understanding of the functions of endogenous melatonin. Tryptophan decarboxylase (TDC) serves as the initial rate-limiting enzyme in the melatonin synthesis pathway. To date, no cucumber TDC gene has been cloned and characterized. In this study, we identified two TDC genes (CsTDC1 and CsTDC2) in the cucumber genome. The subcellular localization analysis indicated that CsTDC1 and CsTDC2 are predominantly localized in the cytoplasm and plasma membrane. Tissue-specific expression analyses revealed that CsTDC1 and CsTDC2 are expressed in both vegetative and reproductive organs. Many cis-elements related to stress, hormone, and light responses as well as development were identified in the CsTDC promoter regions. Furthermore, the expression of CsTDC1 and CsTDC2 was strongly induced by treatments with various abiotic stresses and exogenous hormones. The transient overexpression of CsTDC1 and CsTDC2 in tobacco leaves resulted in increases in the TDC activity and melatonin content, along with improved tolerance of tobacco leaves to salt, drought, and low-temperature stresses. Notably, the overexpression of CsTDC2 had a more pronounced effect than the overexpression of CsTDC1. Accordingly, both CsTDC genes, but especially CsTDC2, may be important for regulating cucumber growth, development, and stress tolerance. The study findings provide a theoretical and experimental basis for future functional analyses of endogenous melatonin in cucumber.

Biosynthesis of melatonin from l-tryptophan by an engineered microbial cell factory

BackgroundThe demand for melatonin is increasing due to its health-promoting bioactivities such as antioxidant and sleep benefits. Although melatonin is present in various organisms, its low content and high extraction cost make it unsustainable. Biosynthesis is a promising alternative method for melatonin production. However, the ectopic production of melatonin in microorganisms is very difficult due to the low or insoluble expression of melatonin synthesis genes. Hence, we aim to explore the biosynthesis of melatonin using Escherichia coli as a cell factory and ways to simultaneously coordinated express genes from different melatonin synthesis pathways.ResultsIn this study, the mXcP4H gene from Xanthomonas campestris, as well as the HsAADC, HsAANAT and HIOMT genes from human melatonin synthesis pathway were optimized and introduced into E. coli via a multi-monocistronic vector. The obtained strain BL7992 successfully synthesized 1.13 mg/L melatonin by utilizing L-tryptophan (l-Trp) as a substrate in a shake flask. It was determined that the rate-limiting enzyme for melatonin synthesis is the arylalkylamine N-acetyltransferase, which is encoded by the HsAANAT gene. Targeted metabolomics analysis of l-Trp revealed that the majority of l-Trp flowed to the indole pathway in BL7992, and knockout of the tnaA gene may be beneficial for increasing melatonin production.ConclusionsA metabolic engineering approach was adopted and melatonin was successfully synthesized from low-cost l-Trp in E. coli. This study provides a rapid and economical strategy for the synthesis of melatonin.

Plant Hormone Signals Mediate Melatonin Synthesis to Enhance Osmotic Stress Tolerance in Watermelon Cells

Melatonin, a multifunctional signaling molecule, has been shown to play a significant role in response to abiotic stress. Several species have been reported to unveil melatonin’s effect on osmotic stress; however, the signal transduction mechanism of phytohormone-mediated melatonin biosynthesis in plant species remains unclear. In this study, although plants can biosynthesize melatonin, the exogenous application of melatonin to watermelon cells can improve cell growth in response to osmotic stress by regulating the antioxidant machinery of cells. Regarding the melatonin synthesis pathway, ClOMT (ClASMT and ClCOMT) is a multi-gene family, and ClSNAT has two members. Both ClOMTs and ClSNATs harbor the cis-elements in their promoter regions responding to various hormones. Among abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), ABA treatment observably upregulated the expression of ClOMTs and ClSNATs, and the accumulation of melatonin with ABA treatment reached a level comparable to that with osmotic stress by mannitol treatment. Furthermore, when hormone biosynthesis inhibitors were added to cells before osmotic stress, the expression of ClOMTs and ClSNATs, as well as melatonin accumulation, were significantly suppressed with the ABA biosynthesis inhibitor. This study demonstrated the crucial role of melatonin biosynthesis in response to osmotic stress via plant hormone signal transduction. It showed that ABA signaling plays a dominant role in melatonin synthesis under osmotic stress.

RP58 knockdown contributes to hypoxia-ischemia-induced pineal dysfunction and circadian rhythm disruption in neonatal rats.

Hypoxia-ischemia (HI) of the brain not only impairs neurodevelopment but also causes pineal gland dysfunction, which leads to circadian rhythm disruption. However, the underlying mechanism of circadian rhythm disruption associated with HI-induced pineal dysfunction remains unknown. The zinc finger protein RP58 is involved in the development and differentiation of nerve cells. In this study, we established an HI model in neonatal rats to investigate the expression of RP58 and its role in pineal dysfunction and circadian rhythm disruption induced by HI. We demonstrated that RP58 was highly expressed in the pineal gland under normal conditions and significantly downregulated in the pineal gland and primary pinealocytes following HI. Knockdown of RP58 decreased the expression of enzymes in the melatonin synthesis pathway (TPH1, ASMT and AANAT) as well as clock genes (CLOCK and BMAL1), and it also reduced the production of melatonin, caused pineal cell injury, and disrupted circadian rhythms in vivo and in vitro. Similarly, HI reduced the expression of melatonin synthesis enzymes (TPH1, ASMT and AANAT) and clock genes (CLOCK and BMAL1) and caused pineal injury and circadian rhythm disruption, which were exacerbated by RP58 knockdown. The detrimental effects of RP58 knockdown on pineal dysfunction and circadian rhythm disruption was reversed by the addition of exogenous melatonin. Furthermore, exogenous melatonin reversed HI-induced pineal dysfunction and circadian rhythm disruption, as reflected by improvements in melatonin production, voluntary activity periods, and activity frequency as well as a diminished decrease in the expression of melatonin synthesis enzymes and clock genes. The present study suggests that RP58 is an endogenous source of protection against pineal dysfunction and circadian rhythm disruption after neonatal HI. This article is protected by copyright. All rights reserved.

Mechanisms of sex differentiation and sex reversal in hermaphrodite fish as revealed by the Epinephelus coioides genome.

Most grouper species are functional protogynous hermaphrodites, but the genetic basis and the molecular mechanisms underlying the regulation of this unique reproductive strategy remain enigmatic. In this study, we report a high-quality chromosome-level genome assembly of the representative orange-spotted grouper (Epinephelus coioides). No duplication or deletion of sex differentiation-related genes was found in the genome, suggesting that sex development in this grouper may be related to changes in regulatory sequences or environmental factors. Transcriptomic analyses showed that aromatase and retinoic acid are probably critical to promoting ovarian fate determination, and follicle-stimulating hormone triggers the female-to-male sex change. Socially controlled sex-change studies revealed that, in sex-changing fish, the brain's response to the social environment may be mediated by activation of the phototransduction cascade and the melatonin synthesis pathway. In summary, our genomic and experimental results provide novel insights into the molecular mechanisms of sex differentiation and sex change in the protogynous groupers.

Blast Exposure Dysregulates Nighttime Melatonin Synthesis and Signaling in the Pineal Gland: A Potential Mechanism of Blast-Induced Sleep Disruptions.

Blast-induced traumatic brain injury (bTBI) frequently results in sleep-wake disturbances. However, limited studies have investigated the molecular signaling mechanisms underlying these sleep disturbances, and potentially efficacious therapies are lacking. We investigated the levels of melatonin and genes involved in melatonin synthesis pathway in the pineal glands of Sprague Dawley rats exposed to single and tightly coupled repeated blasts during the night and daytime. Rats were exposed to single and tightly coupled repeated blasts using an advanced blast simulator. The plasma, cerebrospinal fluid (CSF), and pineal gland were collected at 6 h, 24 h, or 1 month postblast at two different time points: one during the day (1000 h) and one at night (2200 h). Differential expressions of genes involved in pineal melatonin synthesis were quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Plasma and CSF melatonin levels were assessed using a commercial melatonin ELISA kit. The plasma and CSF melatonin levels showed statistically significant decreases at 6 h and 24 h in the blast-exposed rats euthanized in the night (in dim light), with no significant alterations noted in rats euthanized in the morning (daylight) at all three-time points. Blast-exposed rats showed statistically significant decreases in Tph1, Aanat, Asmt, and Mtnr1b mRNA levels, along with increased Tph2 mRNA, in the pineal gland samples collected at night at 6 h and 24 h. No significant changes in the mRNA levels of these genes were noted at 1 month. These findings imply that the melatonin circadian rhythm is disrupted following blast exposure, which may be a factor in the sleep disturbances that blast victims frequently experience.

Approches thérapeutiques des troubles du sommeil et des rythmes chez l’enfant avec TSA

Sleep disturbances are extremely common (40–86%) in children and adolescents, especially those with autism spectrum disorders (ASD) and are often among the first symptoms identified by parents at a very early stage of their child's development. These abnormalities are among the main parental concerns when having a child with ASD and have a significant impact on the quality of life of patients, their parents, and more broadly their siblings. Sleep disorders are essentially abnormalities of the sleep-wake rhythm – primarily sleep onset insomnia or nocturnal awakenings (with difficulty falling back to sleep). These disturbances can be accompanied by other sleep disorders, requiring notably a systematic elimination of the presence of a sleep apnea or restless legs syndrome – to ensure a personalized and efficient therapeutic approach. Physiologically, the determinants of these sleep disorders are poorly understood, even though several studies point to a significant decrease in melatonin synthesis in people with ASD. Melatonin is a hormone that facilitates falling asleep and maintaining sleep and is also involved in the endogenous synchronization of internal biological clocks. However, the causal factors of this decrease in melatonin synthesis are largely unknown, involving to a small extent the genes involved in melatonin synthesis pathway. The treatment of sleep disorders is relatively systematic: after eliminating other specific sleep disorders associated with the complaint of insomnia, as well as other possible associated comorbidities (such as seizures), a global and graduated therapeutic approach must be put in place. This treatment will be non-pharmacological as a first line, then pharmacological as a second line. A number of non-pharmacological treatment strategies for sleep disorders in typically developing children and adolescents, as well as those with ASD, have been shown to be effective. This treatment requires a combination of: 1) parental education to promote sleep development; 2) setting up bedtime rituals adapted to the child's age and particularities; 3) specific behavioral strategies including bedtime fading, gradual extinction and positive reinforcement of adapted behaviors. It is very essential that the parents are accompanied throughout this therapy. Sleep hygiene and behavioral care must also take into consideration the important role of the zeitgebers of sleep-wake rhythms, i.e. the external environmental factors involved in the synchronization of the biological clocks: regular exposure to light at adapted times, regular meal and wake-up times, social activities and times for going to school. The evidence for the effectiveness of behavioral interventions in the treatment of behavioral insomnia in the typical developmental child is strong, since 94% of children show clinically significant improvements in nighttime sleepiness and waking. By contrast, only about 25% of children with ASD are improved by an approach combining sleep hygiene and behavioral therapy. Melatonin has a special and prominent place in the drug management of sleep disorders associated with ASD. Several clinical trials have shown that melatonin is effective in treating sleep disorders in patients with ASD. This work led to the European Medicines Agency (EMA) granting marketing authorization in September 2018 for a sustained-release paediatric melatonin molecule (Slenyto®). This synthetic molecule is a prolonged release melatonin (PRM) which mimics the physiological pharmacokinetic and secretory characteristics of endogenous melatonin, having a very short blood half-life and prolonged secretion for several hours during the night. A recent study evaluated the efficacy and safety of pediatric PRM (mini-tablets) in 125 children, aged 2 to 17.5 years with mainly ASD. After 15 days on placebo, the children were randomized into two parallel groups, PRM or placebo in a double-blind design for 13 weeks. At endpoint, total sleep time was increased by an average of 57.5 minutes on PRM and only 9.14 minutes on placebo (P=0.034). This difference between the two groups was already significant after three weeks of treatment (P=0.006). Sleep latency was also improved in the PRM group (−39.6 minutes) compared to placebo (−12.51 minutes) (P=0.01). Consolidated sleep duration (uninterrupted by awakenings) was improved by 77.9 minutes for the PRM group and only 25.4 minutes for the placebo group (P<0.001). PRM was well tolerated, the most frequent side effects being headache and daytime drowsiness at the same level with PRM or placebo. In addition, the acceptability by the children for swallowing the mini-tablets was excellent (100% compliance). The efficacy and tolerability of PRM was maintained over the medium and long term in the open phase, over a total study duration of 2 years.

Des gènes circadiens à la sémiologie psychiatrique

Psychiatric disorders, such as bipolar disorder and autism spectrum disorders, are highly inherited. Many studies have reported a higher relative risk of developing these disorders than in general population when there is already an affected individual in the family. The heritability of these disorders varies from 70% to 90%. However, the identification of susceptibility genes to these disorders remains difficult, due to their high clinical and etiological heterogeneity. The combination of endophenotypes and environmental factors in genetic studies shed new lights on the biological pathways that are affected in subjects with psychiatric disorders. In particular, sleep disorders and circadian rhythm abnormalities have been frequently reported in these two disorders. Here, we summarize several associations between sleep disorders or circadian rhythm abnormalities and single nucleotide polymorphisms in circadian genes and in genes encoding enzymes of the melatonin synthesis pathway. In particular, we describe studies reporting that polymorphisms in the circadian genes TIMELESS and RORA may increase the risk of developing bipolar disorder by altering circadian rhythms. In addition, we describe studies showing that genetic variations in acetylserotonin methyltransferase (ASMT) identified in patients with psychiatric disorders affect its enzymatic activity. This approach may have therapeutic benefits in bipolar disorders, through the assessment of sleep and circadian rhythms, and a personal management of these alterations.

Melatonin synthesis enzymes interact with ascorbate peroxidase to protect against oxidative stress in cassava

Melatonin is an important indole amine hormone in animals and plants. The enzymes that catalyse melatonin synthesis positively regulate plant stress responses through modulation of the accumulation of reactive oxygen species (ROS). However, the relationship between melatonin biosynthetic enzymes and ROS-scavenging enzymes has not been characterized. In this study, we demonstrate that two enzymes of the melatonin synthesis pathway in Manihot esculenta (MeTDC2 and MeASMT2) directly interact with ascorbate peroxidase (MeAPX2) in both in vitro and in vivo experiments. Notably, in the presence of MeTDC2 and MeASMT2, MeAPX2 showed significantly higher activity and antioxidant capacity than the purified MeAPX2 protein alone. These findings indicate that MeTDC2-MeAPX2 and MeASMT2-MeAPX2 interactions both activate APX activity and increase antioxidant capacity. In addition, the combination of MeTDC2, MeASMT2, and MeAPX2 conferred improved resistance to hydrogen peroxide in Escherichia coli. Moreover, this combination also positively regulates oxidative stress tolerance in cassava. Taken together, these findings not only reveal a direct interaction between MeTDC2, MeASMT2, and MeAPX2, but also highlight the importance of this interaction in regulating redox homoeostasis and stress tolerance in cassava.

CsSNAT positively regulates salt tolerance and growth of cucumber by promoting melatonin biosynthesis

Salt stress is an important limiting factor in agricultural production. Previous studies have shown that exogenous melatonin (N-acetyl-5-methoxytryptamine) promotes plant defense against various abiotic stresses in a concentration dependent manner. It is an efficient and convenient way to improve the resistance of plants by modulating the endogenous melatonin at a moderately high level by utilizing the plant's own regulatory mechanism. Serotonin N-acetyltransferase (SNAT) is a key enzyme in the melatonin synthesis pathway. In this study, CsSNAT overexpression lines and CsSNAT RNA interference (RNAi) lines were generated. The content of endogenous melatonin increased 2-3-fold in overexpression lines (OE-15 and OE-40) and decreased 0.5-0.6-fold in RNAi lines (RNAi-5 and RNAi-7). After three days of treatment with 100 mM NaCl, the overexpression lines exhibited stronger salt tolerance compared to wild type and RNAi lines. The malondialdehyde (MDA) content, relative electrical conductivity (REC), proline content, maximum photochemical efficiency of photosystem II (Fv/Fm), reactive oxygen species (ROS) accumulation, and the activities of antioxidant enzymes showed more significant salt stress injury in the RNAi lines. Under salt stress, there was significantly higher expression of stress resistance-related genes such as CsSOD, CsCAT, CsP5CS1, CsP5CS2, CsPIP, and CsHSP90 in the overexpression lines compared to the wild type and the RNAi lines. Further analysis revealed that overexpression of the CsSNAT gene in cucumber promoted seed germination. The growth of cucumber seedlings of the RNAi lines was severely inhibited. Taken together, our results suggest that CsSNAT positively regulates salt tolerance and growth of cucumber seedlings by promoting melatonin synthesis.

Microorganisms: Producers of Melatonin in Fermented Foods and Beverages.

Melatonin has recently been detected in fermented beverages and foods, in which microorganism metabolism is highly important. The existing literature knowledge discusses the direction for future studies in this review. Evidence shows that many species of microorganisms could synthesize melatonin. However, the actual concentrations of melatonin in fermented foods and beverages range from picograms per milliliter to nanograms per milliliter. Different types of microorganisms, different raw materials, different culture environments, the presence or absence of precursors, high or low alcohol content, and different detection methods are all possible reasons for the huge difference of melatonin levels. Thus far, there have been relatively few studies on the melatonin synthesis pathway microorganisms. Thus, referring to the synthetic pathway of plants and animals, the putative melatonin biosynthesis pathway of microorganisms is presented. It will be significant to discuss whether all species of microorganisms have the capacity to synthesize melatonin and what the biological functions of melatonin are in microorganisms. Melatonin plays a lot of important roles in microorganisms, particularly in enhancing the tolerance of environment stress. Also, the loss of melatonin concentration in commercially available fermented foods and beverages is a ubiquitous trend, and how to solve this problem is a new field to be further explored.

Enhanced melatonin production via aralkylamine N-acetyltransferase overexpression enhances NaCl resistance in transgenic Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

ABSTRACTThe hormone melatonin (MT) is produced by the pineal gland in animals and can also be found in plants and algae. MT regulates not only sleep, reduces the effects of jet lag, and has anti-aging properties in animals, but also eliminates intracellular reactive oxygen species and promotes vegetative growth in plants. However, research on its biological function in microalgae is still rare. The present study involved the construction of the Chlamydomonas reinhardtii expression vector pDBle-AANAT, which harbours the gene of one of the enzymes of the MT synthesis pathway, aralkylamine N-acetyltransferase (AANAT). The AANAT gene was introduced into the genome of the model microalga C. reinhardtii via glass bead transformation. The AANAT transgenic algal strain was identified by resistance screening, culture, and molecular biology methods. Changes in MT content between transformed algal strain and nontransformed algal strains were detected and the antioxidant capacity of the AANAT overexpressing algal strain was analysed under salt stress. The results showed that genetically engineered algae can increase the content of MT in algal cells by overexpressing AANAT. Under salt stress conditions, this can help to improve the enzymatic activity of the antioxidant system of algal cells and reduce membrane damage to counteract the adverse external stress. This enhances the self-protection ability of C. reinhardtii. This study reveals the effects of MT overproduction on the physiological functions of C. reinhardtii. Additionally, the potential functions of MT in the response of C. reinhardtii to stress have been elucidated, thus broadening the functional scope of MT in plants.

A congenic line of the C57BL/6J mouse strain that is proficient in melatonin synthesis.

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from nonfunctional alleles of the genes coding 2 key enzymes of the melatonin synthesis pathway: arylalkylamine-N-acetyltransferase (Aanat) and N-acetylserotonin-O-methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin-proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin-dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin-dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin-proficient B6 congenic line will be widely distributed.

Impact of temperature on sea bass, Dicentrarchus labrax, retina: Fatty acid composition, expression of rhodopsin and enzymes of lipid and melatonin metabolism

Teleost fish are ectothermic vertebrates. Their metabolism, physiology and behavior rely on the external temperature. This study, on the retina of the sea bass Dicentrarchus labrax, reports on the impact of temperature on the fatty acid composition and mRNA abundance of key enzymes of lipid metabolism: fatty acid desaturase-2 (FADS2), fatty acid elongase-5 (ELOVL5), sterol regulatory element-binding protein-1 (SREBP-1), triglyceride lipase and phospholipase A2 (PLA2). We also report on the effects on the photopigment molecule rhodopsin and on enzymes of the melatonin synthesis pathway, namely arylalkylamine N-acetyltransferases 1a and 1b and acetylserotonin methyltransferase. Juvenile fish were placed for 30 days at 18, 23 or 28 °C. At 23 °C, the fatty acid composition of D. labrax retina showed, as generally reported for the retina of other fish species, particularly high amounts of docosahexaenoic (DHA), palmitic and oleic acids. The fatty acids composition was not significantly (P > 0.05) altered between 23 and 28 °C, but did increase at 18 °C compared to 23 and 28 °C. At 18 °C there were noticeable increases in total DHA, ecosapentaenoic, arachidonic, oleic, linoleic, palmitoleic and stearic acids. A negative correlation was found in the abundance of neutral (NL) vs. polar (PL) lipids: 18 °C induced an increase in NL and a decrease in PL, while 28 °C induced higher PL with decreased NL. In NL the changes affected mainly triglycerides. FADS2 and ELOVL5 mRNA abundance decreased from 18° to 28 °C while SREBP-1 and triglyceride lipase mRNA remained stable. Conversely PLA2 mRNA was more abundant at 23 than at 18 and 28 °C. Temperature increased and decreased rhodopsin mRNA abundance, at 28 °C and 18 °C respectively, while there was no effect on mRNA from the melatonin synthesis enzymes. In conclusion the data indicate a temperature induced redistribution of fatty acids among the lipid classes that might affect the physical properties of the plasma membranes as well as functions associated with photoreception or generation of intracellular second messengers. In addition, the results suggest that temperature targets only the proteins and activities of retinal melatonin production. This study opens new lines of investigation related to the role temperature and fatty acids play in fish visual perception. They are relevant in the context of the global warming of seas affecting both the wild and the aquaculture species.

Endophytic Bacterium Pseudomonas fluorescens RG11 May Transform Tryptophan to Melatonin and Promote Endogenous Melatonin Levels in the Roots of Four Grape Cultivars.

Endophytes have been verified to synthesize melatonin in vitro and promote abiotic stress-induced production of endogenous melatonin in grape (Vitis vinifera L.) roots. This study aimed to further characterize the biotransformation of tryptophan to melatonin in the endophytic bacterium Pseudomonas fluorescens RG11 and to investigate its capacity for enhancing endogenous melatonin levels in the roots of different grape cultivars. Using ultra performance liquid chromatography-tandem mass spectrometry combined with 15N double-labeled L-tryptophan as the precursor for melatonin, we detected isotope-labeled 5-hydroxytryptophan, serotonin, N-acetylserotonin, and melatonin, but tryptamine was not detected during the in vitro incubation of P. fluorescens RG11. Furthermore, the production capacity of these four compounds peaked during the exponential growth phase. RG11 colonization increased the endogenous levels of 5-hydroxytryptophan, N-acetylserotonin, and melatonin, but reduced those of tryptamine and serotonin, in the roots of the Red Globe grape cultivar under salt stress conditions. Quantitative real-time PCR revealed that RG11 reduced the transcription of grapevine tryptophan decarboxylase and serotonin N-acetyltransferase genes when compared to the un-inoculated control. These results correlated with decreased reactive oxygen species bursts and cell damage, which were alleviated by RG11 colonization under salt stress conditions. Additionally, RG11 promoted plant growth and enhanced the levels of endogenous melatonin in different grape cultivars. Intraspecific variation in the levels of melatonin precursors was found among four grape cultivars, and the associated root crude extracts appeared to significantly induce RG11 melatonin biosynthesis in vitro. Overall, this study provides useful information that enhances the existing knowledge of a potential melatonin synthesis pathway in rhizobacteria, and it reveals plant–rhizobacterium interactions that affect melatonin biosynthesis in plants subjected to abiotic stress conditions.

Interaction mechanisms of a melatonergic inhibitor in the melatonin synthesis pathway

Acetylserotonin-O-methyltransferase (ASMT) catalyzes the conversion of nor-melatonin to melatonin. Different levels of melatonin have been employed as a trait marker for psychiatric conditions, for this reason ASMT is thought to be an ideal target for developing novel therapeutic agents for the treatment of several mood disorders. In current study, the structural and functional details of the ASMT-B22 complex have been studied using docking and molecular dynamics (MD) simulation. Molecular docking results illustrated that both hydrogen bonding interactions as well as hydrophobic interactions made significant contributions for ligand binding and Arg210 appeared to be the anchor amino acid for the recognition of the receptor. Two hundred nanoseconds MD simulations using AMBER12 were performed to explore the behavior of the ASMT-B22 complex in aqueous solution, focusing on the structural changes induced as a result of inhibitor binding. From root mean square deviation, it was observed that the N-terminal residues endured a conformational change at 11th and 20th ns time scale. The root mean square fluctuation showed high motional flexibility at N-terminal residues because of structural alterations whereas the active site region comprising of a loop was comparatively stable and showed low fluctuations. Radial distribution functions calculated on the major interacting residues of the pocket revealed strengthening of the ASMT-B22 complex indicating the increase in the hydrogen bond interactions. Axial frequency distribution analysis is applied on trajectories for a vivid explanation of hydrogen bonding patterns. Results yielded will be useful in the design of more potent inhibitors of ASMT for normalizing the melatonin levels.

Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone Nematostella vectensis.

BackgroundThe primary hormone of the vertebrate pineal gland, melatonin, has been identified broadly throughout the eukaryotes. While the role for melatonin in cyclic behavior via interactions with the circadian clock has only been reported in vertebrates, comparative research has shown that the transcription-translation loops of the animal circadian clock likely date to the cnidarian-bilaterian ancestor, leaving open significant questions about the evolutionary origin of melatonin signaling in circadian behavior by interacting with the molecular clock.ResultsExpression of melatonin in adult anemones showed peak expression at the end of light period (zeitgeber time (ZT) = 12) when cultured under diel conditions, coinciding with expression of genes and enzyme activity for members of the melatonin synthesis pathway (tryptophan hydroxylase and hydroxyindol-O-methyltransferase), which also showed rhythmic expression. During embryogenesis and juvenile stages, melatonin showed cyclic oscillations in concentration, peaking in midday. Spatial (in situ hybridization) and quantitative (real-time PCR) transcription of clock genes during development of N. vectensis showed these ‘clock’ genes are expressed early in the development, prior to rhythmic oscillations, suggesting functions independent of a function in the circadian clock. Finally, time-course studies revealed that animals transferred from diel conditions to constant darkness lose circadian expression for most of the clock genes within 4 days, which can be reset by melatonin supplementation.ConclusionsOur results support an ancient role for melatonin in the circadian behavior of animals by showing cyclic expression of this hormone under diel conditions, light-dependent oscillations in genes in the melatonin synthesis pathway, and the function of melatonin in initiating expression of circadian clock genes in the cnidarian N. vectensis. The differences in expression melatonin and the circadian clock gene network in the adult stage when compared with developmental stages of N. vectensis suggests new research directions to characterize stage-specific mechanisms of circadian clock function in animals.

Association between ASMT and autistic-like traits in children from a Swedish nationwide cohort

Individuals with autism spectrum disorders often show low levels of melatonin, and it has been suggested that this decrease may be because of the low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin-synthesis pathway. Also, genetic variants in ASMT have been associated with autism, as well as with low ASMT activity and melatonin levels, suggesting that the low ASMT activity observed in autism may partly be because of variations within the ASMT gene. In this study, we present a symptom-based approach to investigate possible associations between ASMT and autistic-like traits in the general population. To this end, continuous measures of autistic-like traits were assessed in a nationally representative twin cohort (n=1771) from Sweden and six single nucleotide polymorphisms (SNPs), and a duplication of exons 2-8 in ASMT were genotyped. Our results show a nominally significant association, in girls, between one single nucleotide polymorphism (rs5949028) in the last intron of ASMT and social interaction impairments. No significant association, however, was observed with traits related to language impairment or restricted and repetitive behavior. In conclusion, our results support the possible involvement of the ASMT gene in autism spectrum disorders, and our finding that only one of the three traits shows association suggests that genetic research may benefit from adopting a symptom-specific approach to identify genes involved in autism psychopathology.

Melatonin synthesis in rice seedlings in vivo is enhanced at high temperatures and under dark conditions due to increased serotonin N‐acetyltransferase and N‐acetylserotonin methyltransferase activities

Temperature and light are important environmental factors for plant growth and development. The final two enzymes in the melatonin synthesis pathway in plants are serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT), which have thermophilic characteristics. Thus, the effects of temperature and light on melatonin synthesis in rice seedlings were investigated. Here, we demonstrated that melatonin levels increased as temperature increased when rice seedlings were exposed to various temperatures for 1hr. Moreover, the relative melatonin levels were higher in the dark. For example, exposure of rice seedlings to 1-hr darkness at 55°C resulted in a melatonin yield of 4.9ng/g fresh weight (fw), compared with 2.95ng/g fw under light conditions. Temperature-dependent melatonin synthesis was closely associated with an increase in both SNAT and ASMT activities, but not with transcript levels of melatonin biosynthetic genes. The daily melatonin levels in field-grown rice plants were unaffected as the positive effect of the relatively high temperature during the day was counteracted by the negative effect of the high light. The opposite effect occurred during the night, in which the positive effect of darkness on melatonin synthesis was counteracted by the negative effect of a low temperature.