Higher Melatonin Concentrations Research Articles

Leveraging 3D-printed microfluidic micromixers for the continuous manufacture of melatonin loaded SNEDDS with enhanced antioxidant activity and skin permeability

Vesicants are chemical warfare agents (CWAs) capable of causing severe skin damage and systemic toxicity. Melatonin, known for its anti-inflammatory and antioxidant properties, can mitigate the effects of these agents. Self-nano-emulsifying drug delivery systems (SNEDDS) containing a high melatonin concentration (5 %, 50 mg/g) were optimized using a quality-by-design approach from biocompatible, non-irritant excipients with a particle size of about 100 nm. The melatonin-loaded SNEDDS showed a 43-fold greater permeability than a conventional melatonin cream. Chemical stability at ambient temperature (25 °C) was maintained for one year. The preparation of optimised melatonin-loaded SNEDDS using a simple mixing method was compared to microfluidic micromixers. Mixing was successfully achieved using a 3D-printed (fused deposition modeling or stereolithography) T-shaped toroidal microfluidic chip (with a channel geometry optimized by computational fluid dynamics), resulting in a scalable, continuous process for the first time with a substantial reduction in preparation time compared to other conventional mixing approaches. No statistically significant differences were observed in the key quality attributes, such as particle size and melatonin loading, between the conventional mixing method in a water bath to reach equilibrium solubility and the use of 3D-printed micromixers. This scalable, continuous, cost-effective approach improves the overall efficiency of SNEDDS production, reduces the cost of quality control for multiple batches, and demonstrates the potential of microfluidic manufacturing with readily customizable 3D-printed micromixers at points of care such as military bases.

Exogenous Application of Melatonin on the Preservation of Physicochemical and Enzymatic Qualities of Pepper Fruit from Chilling Injury

This study investigates the impact of melatonin (MT) treatment at varying concentrations (0, 25, 50, 75, 100 µmol L−1) on the post-harvest quality and shelf life of long green pepper fruits stored under low temperature for 28 days. Every 4 days, pepper fruits were examined for the chilling injury (CI) index, weight loss (WL), respiration rates, firmness, electrolyte leakage (EL), reactive oxygen species (ROS), malondialdehyde (MDA) levels, non-enzymatic antioxidant (NEA) content, antioxidant (AO) enzyme activity (superoxide dismutase (SOD), catalase (CAT), and peroxidases (PODs)), and cellular degrading enzymes (polygalacturonase (PG), pectin methylesterase (PME), phospholipase D (PLD), and lipoxygenase (LOX)). MT-treated samples exhibited delayed and reduced CI stress compared to controls, with higher exogenous MT concentrations (>50 µmol L−1) offering significant (p < 0.05) CI reductions. During storage, WL was notably mitigated by MT treatment in the tested samples compared to control samples. This study also demonstrated that MT-treated pepper fruits effectively decelerated respiration rates and consequently preserved pepper firmness. A higher concentration of MT-treated pepper fruits demonstrated a significantly (p < 0.05) lowered level of ROS and MDA while maintaining membrane stability, as evidenced by reduced EL. MT treatment with increasing concentration increased the levels of glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid (AsA), dehydroascorbate (DHA), and total phenolic content (TPC) in the pepper fruits compared to control and thus significantly (p < 0.05) suppressed the ROS production (superoxide anion (O2•−) and hydrogen peroxide (H2O2) radicals) in the pepper fruits. Furthermore, AO enzymes such as SOD, CAT, and POD were also high in the pepper fruits that were treated with higher concentrations of MT (>50 µmol L−1). Additionally, the activities of cellular degrading enzymes (PG, PME, PLD, and LOX), which are linked to senescence and stress-induced physiological disorders, were also effectively regulated by MT-treated (>75 µmol L−1) pepper fruits. Overall, the application of MT at higher concentrations (>75 µmol L−1) demonstrated substantial benefits in preserving the quality and extending the shelf life of pepper fruits during cold storage.

The Effect of Lemon Juice (Citrus limon L.) Treated with Melatonin on the Health Status and Treatment of K14HPV16 Mice.

Lemon is a fruit rich in antioxidant properties and has several health benefits, namely the reduction of skin edema and anticarcinogenic properties, which are due to its high content of bioactive compounds. Melatonin can improve and preserve the properties of lemon for longer and also has health benefits. The aim of this study was to evaluate the effects of oral administration of lemon juice after melatonin treatment on murinometric parameters of wild-type (WT) mice and transgenic mice carrying human papillomavirus (HPV). Two trials were performed for oral administration of the lemon extract compound: in drinking water and in diet. First of all, lemons were treated by immersion with melatonin at 10 mM. Then, lemons were squeezed, and the juice obtained was freeze-dried and stored to be subsequently added to drinking water or diet, according to the assay. Thus, mice were divided into eight groups in the drink assay (each with n = 5): group 1 (G1, WT, control), group 2 (G2, WT, 1 mL lemon), group 3 (G3, WT, 1.5 mL lemon), group 4 (G4, WT, 2 mL lemon), group 5 (G5, HPV16, control), group 6 (G6, HPV16, 1 mL lemon) group 7 (G6, HPV16, 1.5 mL lemon) and group 8 (G6, HPV16, 2 mL lemon). The diet assay was divided into four groups: group 1 (G1, WT, control), group 2 (G2, WT, 4 mL lemon), group 3 (G3, HPV16, control) and group 4 (G4, HPV16, 4 mL lemon). In the drink assay, the highest concentration of melatonin (308 ng/100 mL) was for groups 4 and 8, while in the food assay, there was only one concentration of melatonin (9.96 ng/g) for groups 2 and 4. Both trials lasted 30 days. During this time, body weight, food and water were recorded. Afterward, they were sacrificed, and samples were collected for different analyses. At the concentrations used, the lemon juice with melatonin had no adverse effects on the animals' health and showed a positive outcome in modifying weight gain and enhancing antioxidant activity in mice. Moreover, a reduction in the incidence of histological lesions was observed in treated animals. Further research is needed to better understand the effects of lemon extract on health and treatment outcomes in this animal model.

Influence of Melatonin Coating on Physiochemical Qualities and Enzymatic Activities in Banana Pericarp under Cold Storage

Banana pericarp is highly susceptible to chilling injury (CI), negatively affecting its quality and shelf life. Melatonin (MT), a plant tryptophan derivative, has shown promising effects in mitigating CI and related physiological disorders in tropical and subtropical fruits. This study investigated the efficacy of MT at different concentrations (0, 50, and 100 µM/L) in the coating of banana pericarp to control CI and physicochemical degradation during storage at 7 °C for five days, and on each day, fruits were assessed for quality. The MT100 treatment significantly (p < 0.05) mitigated the severity of the CI index, electrolytic leakage (EL), and malondialdehyde (MDA) levels. Reactive oxygen species (ROS) levels were substantially higher in control samples, whereas MT treatments notably suppressed their increase. Glutathione (GSH) and ascorbic acid (AA) levels were elevated in those banana pericarps treated with higher MT concentrations. Although total phenolic content (TPC) and total carotenoid contents (TCC) were increased in MT-treated samples, the MT concentrations did not significantly affect them. The level of phenolic compounds, such as gallic acid (GA), chlorogenic acid (CA), quinic acid (QA), protocatechuic acid (PA), and catechin (CC), exhibited continuous growth during the storage period, with the highest levels found in MT100-treated samples. Activities of enzymes such as lipoxygenase (LOX), phospholipase D (PLD), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) were significantly (p < 0.05) higher in control samples and rose continuously over time, yet were effectively reduced in MT-treated pericarps. This study shows that applying a higher MT coating (100 µM/L) to bananas is an effective post-harvest strategy to considerably lower the incidence of CI and associated losses.

FEATURES OF RELATIONS OF MELATONIN WITH THE STATE OF INTRACELLULAR REGULATORSOF THE FUNCTIONAL ACTIVITY OF WHOLE BLOOD MONONUCLEAR CELLS IN CORONARY HEART DISEASE

Introduction. Coronary heart disease (CHD), leading among the causes of death in adulthood and old age, is an urgent medical and social problem. The pathogenesis of most forms of coronary heart disease is based on stenosing atherosclerosis of the coronary arteries, which develops against the background of dyslipidemia and arterial hypertension and is accompanied by the activation of immunocompetent cells (ICCs) of the vascular wall with the development of a subclinical inflammatory reaction, as well as the production of pro-inflammatory factors such as interleukins, chemokines, growth factors and etc. In turn, ICC activity is determined by the state of their intracellular molecular cascades, which transmit signals into the cell and ensure its reactivity to various external stimuli, such as mitogens, cytokines, pathogen components, etcIt has been shown that the central nervous system plays an important role in the regulation of ICC activity due to the production of neurohumoral molecules, such as melatonin, endorphin, sero-tonin, etc., which ensure the coordination of immune responses and their control by the central nervous system. The aim of this study was to study the relationship between melatonin production and intracellular factors that regulate the pro-inflammatory activi-ty of whole blood mononuclear cells and their metabolism in patients with coronary artery disease. Material and methods. As part of the cohort study, 58 patients of both sexes with coronary artery disease aged 49 to 67 years and 20 practically healthy individuals of both sexes were examined. In accordance with the purpose of the study, the concentration of focal adhesion protein kinase (FAK), 5'AMP-activated protein kinase (AMPK), AKT1 protein kinase, signal transducers and transcription activators (STAT) was determined in nuclear cy-toplasmic lysates of whole blood mononuclear cells: STAT3, STAT5A and STAT6, c-Jun N-terminal protein kinase 1 and 2 isoforms (JNK), mitogen-activated protein kinase p38 (p38), extracellular growth kinase 1 and 2 isoforms (ERK), Janus kinase type 2 (JAK2), nuclear transcription factor NF -kB, caspase-1, cyclooxygenase-2 (COX-2), p70-S6K1 protein kinase, p53, p27, p21 proteins. In addition, the concentration of cyclic adenosine monophos-phate (cAMP) and cyclic guanosine monophosphate (cGMP) was determined in cell supernatants. Melatonin concentration was determined in blood se-rum. The material for the study was venous blood taken from the cubital vein in the morning from 6.00 to 6.15. Results. The analysis showed that in patients with coronary artery disease, in comparison with practically healthy individuals, in MNCs of whole blood, there was an increased level of protein kinases FAK, AKT, JNK, ERK, p70-S6K1, factor STAT6, protein p21, against which there was a decrease in the content of STAT3, STAT5A, JAK2, transcription factor NF-kB and caspase-1. These changes were accompanied by increased levels of cGMP and cAMP. Against this background, a decrease in the content of factors was revealed in the MNC. A high concentration of melatonin in patients with CAD was as-sociated with a decrease in the content of protein kinases AMPK, AKT, Jak2, ERK1, protein p21, caspase-1, and cAMP in MNCs, which was observed against the background of an increase in the level of protein p27 and nuclear factor NF-kB. The results of the correlation analysis indicate a different na-ture of the relationship between the level of melatonin and such factors as caspase-1, protein kinases ERK, JAK2, as well as the transcription factor NF-kB and p21 protein, depending on the characteristics of melatonin production in patients with coronary artery disease. Conclusions. In patients with coronary artery disease, melatonin exhibits a modulating effect on the energy balance of ICCs and their metabolism, helps limit pro-inflammatory activity by limiting the functional activity of MAPK/SAPK signaling pathways in MNCs.

Effects of exogenous melatonin on the osmotic regulation and antioxidant capacity of Ginkgo biloba seedlings under salt stress.

We investigated the effects of exogenous melatonin on the osmotic regulation and antioxidant capacity of 4-year-old Ginkgo biloba seedlings under salt stress. There were three treatments, with low (50 mmol·L-1), medium (100 mmol·L-1), and high (200 mmol·L-1) NaCl stress. Leaves were sprayed and the soil was watered with melatonin solution (0, 0.02, 0.1, 0.5 mmol·L-1). The results showed that saline stress significantly inhibited the osmoregulation and antioxidant capacities of G. biloba seedlings. Application of exogenous melatonin at appropriate concentrations (0.02, 0.1 mmol·L-1) under salt stress could promote plant growth, reduce the rate of electrolyte leakage, decrease the content of flavonoids and malonic dialdehyde, and enhance peroxidase and superoxide dismutase activities in leaves. High concentration (0.5 mmol·L-1) of exogenous melatonin would aggravate the oxidative and osmotic stresses. The 0.02 and 0.1 mmol·L-1 exogenous melatonin alleviated osmotic stress and oxidative stress in G. biloba seedlings under salt stress, while the 0.02 mmol·L-1 exogenous melatonin treatment had the best effect on NaCl stress alleviation. Ground diameter, branch width, branch length, electrolyte leakage rate, superoxide dismutase activity, and flavonoids content could be used as the key indices for rapid identification of the degree of salt stress in G. biloba seedlings.

Effects of Exogenous Melatonin on the Growth and Physiological Characteristics of Ginkgo biloba L. under Salinity Stress Conditions

Ginkgo (Ginkgo biloba L.) is a cherished relic among plants, commonly planted as a street tree. However, it faces cultivation challenges due to escalating soil salinization and widespread snowmelt application. Therefore, this study used 4-year-old Ginkgo seedlings to investigate how exogenous melatonin at varying concentrations affects seedling growth and physiology under salinity stress. The results revealed that appropriate melatonin concentrations (0.02, 0.1 mmol·L−1) significantly mitigated leaf yellowing under different NaCl stress levels. Furthermore, they increased ground diameter, current-year branch growth, relative water concentration, free proline, and soluble sugars in leaves. Melatonin also reduced electrolyte exudation rates, flavonoids, and malonic dialdehyde concentration, while enhancing peroxidase and superoxide dismutase activities. This led to reduced chlorophyll content, photosynthetic rate, stomatal conductance, and transpiration rate, stabilizing intercellular CO2 concentration, preserving photosynthetic structures, and enhancing photosynthetic rates. Additionally, the decline in the photosynthetic electron transport rate, the effective photochemical quantum yield of PSII, and the potential efficiency of primary conversion of light energy of PSII was alleviated. Minimal fluorescence and the non-photochemical quenching coefficient also improved. However, high melatonin concentration (0.5 mmol·L−1) exacerbated salinity stress. After analyzing composite scores, the 0.02 mmol·L−1 melatonin treatment was most effective in alleviating NaCl stress, while the 0.5 mmol·L−1 treatment intensified physiological stress under 200 mmol·L−1 NaCl stress. Principal component analysis and correlation analysis identified seven physiological indicators (photosynthetic rate, transpiration rate, photosynthetic electron transport rate, minimal fluorescence, superoxide dismutase, free proline, and chlorophyll a) and three growth indicators (ground diameter, branch length, and current-year branch thickness) as key markers for rapid salinity stress assessment in Ginkgo. These findings are crucial for addressing challenges associated with snowmelt’s impact on roadside Ginkgo trees, expanding planting areas, and breeding exceptional salt-tolerant Ginkgo varieties.

Investigation of Melatonin Incorporated CMC-Gelatin Based Edible Coating on the Alleviation of Chilling Injury Induced Pericarp Browning in Longkong.

Longkong (Aglaia dookkoo Griff.) fruit is prone to rapid pericarp browning and shortened shelf life (<7 days) under prolonged low-temperature storage. This study investigates the effect of an edible coating, comprising carboxymethyl cellulose (CMC) and gelatin in a fixed 3:1 ratio, integrated with various concentrations of melatonin (MT) (0.4, 0.8, and 1.2 mM/L) to mitigate chilling injury in longkong fruit. Coated longkong fruits were stored at 13 °C with 90% relative humidity for 18 days and underwent physicochemical evaluations every three days. Samples coated with CMC-Gel without MT and uncoated fruits were served as controls. The findings indicated that the CMC-Gel-MT coating significantly mitigated pericarp browning, chilling injury, weight loss, and respiration rate increase under extended cold storage conditions. High concentrations of MT (≥0.8 mM/L) in the coating notably inhibited the activities of cellular degrading enzymes such as lipoxygenase and phospholipase D. This inhibition contributed to reduced membrane permeability, lower reactive oxygen species accumulation (H2O2, OH-, O2-), and decreased malondialdehyde levels in the longkong pericarp. Furthermore, the CMC-Gel-MT coating increased the activity of phenylalanine ammonia lyase, leading to an enhancement in phenolic content. Consequently, it improved the fruit's ability to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,20-azino-di-3-ethylbenzthiazoline sulfonic acid) radicals. Control samples exhibited high levels of pericarp browning-related enzymes (polyphenol oxidase, peroxidase), whereas CMC-Gel-MT-coated fruits, particularly at higher MT concentrations, showed significant reductions in those enzyme activities. In conclusion, incorporating high concentrations of MT in a CMC-Gel-based edible coating is a promising alternative for mitigating chilling injury in longkong fruit.

Exogenous Melatonin Regulates Superoxide Dismutase Gene Expression and Enzymatic Activity in Response to Pathogenic Fungi in Soybean Glycine max (L.) Merrill

Melatonin is a pleiotropic molecule known to play a key role in plant growth and development, as well as biotic and/or abiotic stress responses. However, melatonin specific effects on the plant defense system, particularly SOD activity are still an area of ongoing research. The results indicated a clear effect of the applied melatonin concentration in shaping the entire response to the biotic oxidative stress resulted from the two pathogenic fungi Rhizoctonia solani and Macrophomina phaseolina. The higher concentration of melatonin had a greater effect on both gene expression and SOD enzymatic activity, which indicates the melatonin importance in stimulating the defense system of soybean plants through which soybean plants try to manage the excessive ROS accumulation in response to pathogenic fungi. The different varieties showed different ability to cope with biotic stress of pathogenic fungi, especially the antioxidants enzymatic activity. Al-Shaimaa variety proved to have the most effective SOD gene expression and enzymatic activity compared to the other two Senaya22 and Lee74 soybean varieties. Melatonin concentrations in its maximum level (300 μM), was more effective in promoting the antioxidant activity in term of gene expression and enzymatic activity of SOD. Interestingly, R. solani had a greater effect on the antioxidant defense system, as it resulted in higher levels of SOD gene expression and enzymatic activity in the three soybean varieties. Further investigation maybe required to better understand the melatonin role in inducing plant antioxidant system, thus the possible protective mechanisms against plant pathogens.

Foliar application of either melatonin or sodium nitpoprusside regulates the antioxidant status, and the morpho-physiological attributes and essential oil production in sage (Salvia officinalis L.) under salinity stress

Soil salinity is one of the most severe abiotic stress factors affecting crop growth and yield. Among the molecules used to mitigate the adverse effects of salt, melatonin (MT) and the nitric oxide donor sodium nitroprusside (SNP) played a crucial role in mediating plant responses to salt stress. However, the molecules are worthy of further consideration and investigation with regard to the secondary metabolism of plants suffering from salt stress. Herein, the potential role of MT and SNP in alleviating/buffering the negative effects of salt stress on sage (Salvia officinalis L.) seedlings was investigated. In this context, MT (0, 50 µM, and 100 µM) and SNP (0, 50 µM, and 100 µM) were applied individually. The interactive effects of each molecule with salt stress (50 and 100 mM NaCl) were assessed using a range of morpho-physiological, biochemical and analytical parameters of sage. The results of the study showed that high salinity (100 mM NaCl) critically reduced growth and photosynthetic traits and increased oxidative stress damage parameters. On the other hand, high concentrations (100 µM) of MT or SNP treatments significantly improved growth, enhanced photosynthetic traits and mitigated oxidative stress damage parameters. For instance, individual treatments of both MT and SNP enhanced tolerance of sage against salinity stress by increasing relative water content, proline, total carbohydrates, total phenolics and flavonoid content, and the antioxidant enzymes and DPPH scavenging activities. Essential oil yield and individual essential oil compounds were also increased by MT and SNP. Overall, these molecules can be considered as potential protective agents against salinity stress in sage seedlings.

Regulation of Adipose-Derived Stem Cell Activity by Melatonin Receptors in Terms of Viability and Osteogenic Differentiation.

Melatonin is a hormone secreted mainly by the pineal gland and acts through the Mel1A and Mel1B receptors. Among other actions, melatonin significantly increases osteogenesis during bone regeneration. Human adipose-derived mesenchymal stem cells (ADSCs) are also known to have the potential to differentiate into osteoblast-like cells; however, inefficient culturing due to the loss of properties over time or low cell survival rates on scaffolds is a limitation. Improving the process of ADSC expansion in vitro is crucial for its further successful use in bone regeneration. This study aimed to assess the effect of melatonin on ADSC characteristics, including osteogenicity. We assessed ADSC viability at different melatonin concentrations as well as the effect on its receptor inhibitors (luzindole or 4-P-PDOT). Moreover, we analyzed the ADSC phenotype, apoptosis, cell cycle, and expression of MTNR1A and MTNR1B receptors, and its potential for osteogenic differentiation. We found that ADSCs treated with melatonin at a concentration of 100 µM had a higher viability compared to those treated at higher melatonin concentrations. Melatonin did not change the phenotype of ADSCs or induce apoptosis and it promoted the activity of some osteogenesis-related genes. We concluded that melatonin is safe, non-toxic to normal ADSCs in vitro, and can be used in regenerative medicine at low doses (100 μM) to improve cell viability without negatively affecting the osteogenic potential of these cells.

Efficacy, mechanism, and safety of melatonin-loaded on thermosensitive nanogels for rabbit VX2 tumor embolization: A novel design.

Transarterial chemoembolization (TACE) has been widely used for hepatocellular carcinoma.Reducing hypoxia in the tumor microenvironment after TACE remains a challenge as tumor progression is common in post-TACE patients due to the hypoxic tumor microenvironment. In this study, melatonin loaded on p(N-isopropyl-acrylamide-co-butyl methylacrylate) (PIB-M) was used for tumor embolism. Two types of human hepatoma cell lines were used to explore the mechanism by which melatonin prevents the growth and metastasis of cancer cells in vitro. A VX2 rabbit tumor model was used to evaluate the efficacy, mechanism, and safety of PIB-M in vivo. We found that under hypoxic condition, melatonin could inhibit tumor cell proliferation and migration by targeting hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGF-A) in vitro. In vivo, PIB-M inhibited tumor growth and metastasis in rabbit VX2 tumors by promoting apoptosis of tumor cells and targeting related angiogenic proteins and vascular permeability proteins. A high concentration of melatonin in the PIB-M group could be maintained in tumor tissue for 72 h after embolization. The liver and kidney functions were most damaged on the first day but recovered to normal on the seventh day after embolization in the PIB-M group. This novel method may open avenues for reduction of tumor growth and metastasis after TACE and is efficacy and safety, which may be used for treatment for other solid tumors and clinical translation.

A Pilot Study to Evaluate the Potential of Melatonin Implants to Control Cyclicity in the Bitch.

In short-day breeders such as the sheep, melatonin stimulates oestrus activity; in contrast, a high serum concentration of melatonin inhibits oestrus in long-day breeders such as the cat. Therefore, implants with melatonin have been used to suppress or induce oestrus depending on the species. The aim of this pilot study was to evaluate if melatonin could be an alternative to control the reproductive cycle in the bitch. Nine beagle bitches were observed for three oestrus cycles. Five beagle bitches were treated with 18 mg melatonin implants on average 27 days before the next expected oestrus based on the previous interoestrus interval. Four bitches served as untreated controls. Blood samples for evaluation of serum melatonin were collected at the time of assignment to treatment or control group and 1-4 weeks thereafter. Clinical signs and vaginal smears were used to follow the cycle. Melatonin varied significantly with bitch (p < 0.05) but not with treatment. Treatment did not affect the interoestrus interval (p > 0.05). In conclusion, treatment with 18 mg melatonin implants approximately one month before expected oestrus is not likely to be a useful method to control cyclicity in the bitch. It is still not known if melatonin is involved in regulation of the oestrus cycle in the domestic dog.

Melatonin regulates gene expressions through activating auxin synthesis and signaling pathways.

Both melatonin and indole-3-acetic acid (IAA) are derived from tryptophan. And the most interesting and unsolved puzzle in melatonin research is that what is the relationship between melatonin and auxin? In this study, we performed transcriptome analysis with a time series method to disclose the connection of the two metabolites in soybean. Our results reveal that melatonin and IAA treatments cause substantial overlaps in gene expression changes. Common genes of melatonin and IAA treatments could be sorted into clusters with very similar expression tendency. A KEGG assay showed that exogenous applied melatonin enriched differentially expressed genes in auxin biosynthesis and signaling pathways. For details, melatonin up-regulates several YUCCA genes which participate in auxin biosynthesis; melatonin also enhances expression levels of auxin receptor coding genes, such as TIR1, AFB3 and AFB5; dozens of genes involved in auxin transport, such as AUXI and PIN, are regulated by melatonin similarly as by auxin; auxin-responsive genes, such as IAA, ARF, GH3 and SAUR-like genes, intensively respond to melatonin as well as to auxin. A DR5 promoter mediated GUS staining assay showed that low concentration of melatonin could induce auxin biosynthesis in a dosage manner, whereas high concentration of melatonin would eliminate such effect. At last, gene ontology (GO) analysis suggests that melatonin treatment has similar characteristics as auxin treatment in many processes. However, the two molecules still keep their own features respectively. For example, melatonin takes part in stress responses, while IAA treatment enriches the GO terms that related to cell growth. Taken together, exogenous applied melatonin, if not exceeds the appropriate concentration, could promote auxin responses range from biosynthesis to signaling transduction. Thus, our research is a key part to explain the auxin-like roles of melatonin in regulating plant growth.

Insights into melatonin-induced photosynthetic electron transport under low-temperature stress in cucumber.

In this study, the differences in chlorophyll fluorescence transient (OJIP) and modulated 820 nm reflection (MR820) of cucumber leaves were probed to demonstrate an insight into the precise influence of melatonin (MT) on cucumber photosystems under low temperature stress. We pre-treated cucumber seedlings with different levels of MT (0, 25, 50, 100, 200, and 400 μmol · L-1) before imposing low temperature stress (10 °C/6 °C). The results indicated that moderate concentrations of MT had a positive effect on the growth of low temperature-stressed cucumber seedlings. Under low temperature stress conditions, 100 μmol · L-1 (MT 100) improved the performance of the active photosystem II (PSII) reaction centers (PIabs), the oxygen evolving complex activity (OEC centers) and electron transport between PSII and PSI, mainly by decreasing the L-band, K-band, and G-band, but showed differences with different duration of low temperature stress. In addition, these indicators related to quantum yield and energy flux of PSII regulated by MT indicated that MT (MT 100) effectively protected the electron transport and energy distribution in the photosystem. According to the results of WO-I ≥ 1 and MR820 signals, MT also affected PSI activity. MT 100 decreased the minimal value of MR/MRO and the oxidation rate of plastocyanin (PC) and PSI reaction center (P700) (Vox ), while increased △MRslow/MRO and deoxidation rates of PC+ and P700 + (Vred ). The loss of the slow phase of MT 200 and MT 400-treated plants in the MR820 kinetics was due to the complete prevention of electron movement from PSII to re-reduce the PC+ and P700 +. These results suggest that appropriate MT concentration (100 μmol · L-1) can improve the photosynthetic performance of PS II and electron transport from primary quinone electron acceptor (QA) to secondary quinone electron acceptor (QB), promote the balance of energy distribution, strengthen the connectivity of PSI and PSII, improve the electron flow of PSII via QA to PC+ and P700 + from reaching PSI by regulating multiple sites of electron transport chain in photosynthesis, and increase the pool size and reduction rates of PSI in low temperature-stressed cucumber plants, All these modifications by MT 100 treatment promoted the photosynthetic electron transfer smoothly, and further restored the cucumber plant growth under low temperature stress. Therefore, we conclude that spraying MT at an appropriate concentration is beneficial for protecting the photosynthetic electron transport chain, while spraying high concentrations of MT has a negative effect on regulating the low temperature tolerance in cucumber.

Postharvest Exogenous Melatonin Treatment of Table Grape Berry Enhances Quality and Maintains Bioactive Compounds during Refrigerated Storage

Table grape berries are classified as a perishable crop that deteriorates quickly after harvest. The application of melatonin after harvest was found to be effective for retarding senescence and slowing ripening. In the current study, we tested the influence of two melatonin concentrations (50 and 100 µmol) as a postharvest application on quality, bioactive compounds, and enzyme activities of grape berries cv “Crimson” stored at 0 ± 1 °C and 90% relative humidity (RH) for 35 days. Our results indicated that melatonin application extends the shelf-life of berries by reducing weight loss and maintaining total soluble solids (TSS), titratable acidity (TA), berry adherence strength, and firmness. Melatonin treatment also reduced pectin methyl esterase (PME) and polygalactouranase (PG) enzyme activities compared to the control. Moreover, O2•− and H2O2 rates in berries were reduced by high melatonin concentration. Moreover, peroxidase (POD) and catalase (CAT) enzyme activities were increased by melatonin application. Our findings suggested using melatonin postharvest to increase shelf life and maintain quality attributes during refrigerated storage, which could be advantageous on a large scale.

Genome-wide identification, characterization of Serotonin N-acetyltransferase and deciphering its importance under development, biotic and abiotic stress in soybean

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme involved in plant melatonin biosynthesis. Identifying its expression under development and stress will reveal the regulatory role in the soybean. To identify and characterize SNAT, we employed genome-wide analysis, gene structure, cis-acting elements, expression, and enzyme activity. We identified seven putative genes by genome-wide analysis and found chloroplast signal peptides in three GmSNATs. To elucidate GmSNATs role, expression datasets of more than a hundred samples related to circadian rhythm, developmental stages, and stress conditions were analysed. Notably, the expression of GmSNAT1 did not show significant expression during biotic and abiotic stress. The GmSNAT1 sequence showed 67.8 and 72.2 % similarities with OsSNAT and AtSNAT, respectively. The Km and Vmax of the purified recombinant GmSNAT1 were 657 μM and 3780 pmol/min/mg, respectively. To further understand the GmSNAT1 role, we supplemented different concentrations of serotonin and melatonin to in-vitro cultures and seed priming. These studies revealed that the GmSNAT1 expression was significantly up-regulated at higher concentrations of serotonin and down-regulated at higher melatonin concentrations. We speculate that a high concentration of melatonin during abiotic, biotic stress, and in-vitro cultures are responsible for regulating GmSNAT1 expression, which may regulate them at the enzyme level during stress in soybean.

Effects of Melatonin Priming on Suaeda corniculata Seed Germination, Antioxidant Defense, and Reserve Mobilization: Implications for Salinized Wetland Restoration

Melatonin priming has been widely reported to positively affect seed germination under abiotic stresses. However, there is still a gap in knowledge on how melatonin priming impacts the seed germination and physiological change of wetland plant species. We assessed the effects of different melatonin concentrations on germination characteristics, antioxidant defense, and reserve mobilization of Suaeda corniculata seeds. Priming of S. corniculata seeds with 50 μM melatonin significantly improved the germination rate, germination speed, germination index, superoxide dismutase and peroxidase activity, and soluble sugar content as compared with the control, and effectively reduced the malondialdehyde content, promoted starch, soluble protein, and fat mobilization. However, the stress tolerance ability of S. corniculata seeds was reduced by high melatonin concentration. The structural equation model indicated that the melatonin priming directly affects the seed germination, while also indirectly regulating the antioxidant defense system and reserve mobilization. In conclusion, melatonin priming affects the S. corniculata seed germination under salinization stress in a concentration-dependent manner via both direct and indirect regulatory pathways. Insights into these aspects will advance our understanding of how melatonin priming affects S. corniculata seed germination and provide invaluable information and technical support for the restoration of salinized wetlands in the Momoge National Nature Reserve.

Identification and Engineering of Transporters for Efficient Melatonin Production in Escherichia coli.

Transporter discovery and engineering play an important role in cell factory development. Decreasing the intracellular concentration of the product reduces product inhibition and/or toxicity. Lowering intracellular concentrations is especially beneficial for achieving a robust strain at high titers. However, the identification of transporters for xenobiotic chemicals in the host strain is challenging. Here we present a high-throughput workflow to discover Escherichia coli transporters responsible for the efflux of the inhibitory xenobiotic compound melatonin. We took advantage of the Keio collection and screened about 400 transporter knockouts in the presence of a high concentration of melatonin. We found five transporters that when knocked out showed decreased tolerance to melatonin, indicating they are exporters of melatonin. We overexpressed these five genes individually in the production strain and found that one of them, yhjV, encoding a transporter with unknown substrates, resulted in a 27% titer increase in cultivation mimicking fed-batch fermentation. This study demonstrates how microbial cell factories can be improved through transporter identification and engineering. Further, these results lay the foundation for the scale-up of melatonin production in E. coli.

The effect of melatonin on the mouse ameloblast-lineage cell line ALCs

Melatonin plays a critical role in promoting the proliferation of osteoblasts and the growth and development of dental papilla cells. However, the effect and mechanism of melatonin on the growth and development of ALCs still need to be explored. CCK8 assay was used for the evaluation of cell numbers. qRT-PCR was used to identify the differentially expressed genes in ALCs after melatonin treatment. The number and morphology of ALCs were investigated by confocal microscopy. Alkaline phosphatase assay and Alizarin red S staining were used for measuring mineralization. Then, we focused on observing the crucial factors of the signaling pathway by RNA-seq and qRT-PCR. Melatonin limited the cell number of ALCs in a dose-dependent manner and promoted the production of actin fibers. A high concentration of melatonin significantly promoted the mRNA levels of enamel matrix proteins and the formation of mineralized nodules. RNA-seq data showed that Wnt signaling pathway may be involved in the differentiation of ALCs under the influence of melatonin. This study suggests that melatonin plays a regulatory role in the cell number, differentiation, and mineralization of the ALCs, and then shows the relationship between the Wnt signaling pathway with the ALCs under melatonin.