Comparative genomics of N-acetyl-5-methoxytryptamine members in four Prunus species with insights into bud dormancy and abiotic stress responses in Prunus avium.

KEY MESSAGE: This study provides novel insights into the evolution, diversification, and functions of melatonin biosynthesis genes in Prunus species, highlighting their potential role in regulating bud dormancy and abiotic stresses. The biosynthesis of melatonin (MEL) in plants is primarily governed by enzymatic reactions involving key enzymes such as serotonin N-acetyltransferase (SNAT), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT) and tryptophan decarboxylase (TDC). In this study, we analyzed Melatonin genes in four Prunus species such as Prunus avium (Pavi), Prunus pusilliflora (Ppus), Prunus serulata (Pser), and Prunus persica (Pper) based on comparative genomics approach. Among the four Prunus species, a total of 29 TDCs, 998 T5Hs, 16 SNATs, and 115 ASMTs within the genome of four Prunus genomes. A thorough investigation of melatonin-related genes was carried out using systematic biological methods and comparative genomics. Through phylogenetic analysis, orthologous clusters, Go enrichment, syntenic relationship, and gene duplication analysis, we discovered both similarities and variations in Melatonin genes among these Prunus species. Additionally, our study revealed the existence of unique subgroup members in the Melatonin genes of these species, which were distinct from those found in Arabidopsis genes. Furthermore, the transcriptomic expression analysis revealed the potential significance of melatonin genes in bud dormancy regulation and abiotic stresses. Our extensive results offer valuable perspectives on the evolutionary patterns, intricate expansion, and functions of PavMEL genes. Given their promising attributes, PavTDCs, PavT5H, PavNAT, and three PavASMT genes warrant in-depth exploration as prime candidates for manipulating dormancy in sweet cherry. This was done to lay the foundation for future explorations into the structural and functional aspects of these factors in Prunus species. This study offers significant insights into the functions of ASMT, SNAT, T5H, and TDC genes and sheds light on their roles in Prunus avium. Moreover, it established a robust foundation for further exploration functional characterization of melatonin genes in fruit species.

α-Synuclein reduces acetylserotonin O-methyltransferase mediated melatonin biosynthesis by microtubule-associated protein 1 light chain 3 beta-related degradation pathway.

Previous studies have demonstrated that α-synuclein (α-SYN) is closely associated with rapid eye movement sleep behavior disorder (RBD) related to several neurodegenerative disorders. However, the exact molecular mechanisms are still rarely investigated. In the present study, we found that in the α-SYNA53T induced RBD-like behavior mouse model, the melatonin level in the plasma and pineal gland were significantly decreased. To elucidate the underlying mechanism of α-SYN-induced melatonin reduction, we investigated the effect of α-SYN in melatonin biosynthesis. Our findings showed that α-SYN reduced the level and activity of melatonin synthesis enzyme acetylserotonin O-methyltransferase (ASMT) in the pineal gland and in the cell cultures. In addition, we found that microtubule-associated protein 1 light chain 3 beta (LC3B) as an important autophagy adapter is involved in the degradation of ASMT. Immunoprecipitation assays revealed that α-SYN increases the binding between LC3B and ASMT, leading to ASMT degradation and a consequent reduction in melatonin biosynthesis. Collectively, our results demonstrate the molecular mechanisms of α-SYN in melatonin biosynthesis, indicating that melatonin is an important molecule involved in the α-SYN-associated RBD-like behaviors, which may provide a potential therapeutic target for RBD of Parkinson's disease.

ASMT determines gut microbiota and increases neurobehavioral adaptability to exercise in female mice.

N-acetylserotonin O-methyltransferase (ASMT) is responsible for melatonin biosynthesis. The Asmt gene is located on the X chromosome, and its genetic polymorphism is associated with depression in humans. However, the underlying mechanism remains unclear. Here, we use CRISPR/Cas9 to delete 20 bp of exon 2 of Asmt, and construct C57BL/6J mouse strain with Asmt frameshift mutation (Asmtft/ft). We show that female Asmtft/ft mice exhibit anxiety- and depression-like behaviors, accompanied by an obvious structural remodeling of gut microbiota. These behavioral abnormalities are not observed in male. Moreover, female Asmtft/ft mice show a lower neurobehavioral adaptability to exercise, while wild-type shows a "higher resilience". Cross-sectional and longitudinal analysis indicates that the structure of gut microbiota in Asmtft/ft mice is less affected by exercise. These results suggests that Asmt maintains the plasticity of gut microbiota in female, thereby enhancing the neurobehavioral adaptability to exercise.

Unraveling the regulatory role of MYC2 on ASMT gene expression in wheat: Implications for melatonin biosynthesis and drought tolerance.

Recognized for its multifaceted functions, melatonin is a hormone found in both animals and plants. In the plant kingdom, it plays diverse roles, regulating growth, development, and stress responses. Notably, melatonin demonstrates its significance by mitigating the effects of abiotic stresses like drought. However, understanding the precise regulatory mechanisms controlling melatonin biosynthesis genes, especially during monocots' response to stresses, requires further exploration. Seeking to understand the molecular basis of drought stress tolerance in wheat, we analyzed RNA-Seq libraries of wheat exposed to drought stress using bioinformatics methods. In light of our findings, we identified that the Myelocytomatosis oncogenes 2 (MYC2) transcription factor is a hub gene upstream of a main melatonin biosynthesis gene, N-acetylserotonin methyltransferase (ASMT), in the wheat drought response-gene network. Promoter analysis of the ASMT gene suggested that it might be a target gene of MYC2. We conducted a set of molecular and physiochemical assays along with robust machine learning approaches to elevate those findings further. MYC2 and ASMT were co-regulated under Jasmonate, drought, and a combination of them in the leaf tissues of wheat was detected. A meaningful correlation was observed among gene expression profiles, melatonin contents, photosynthetic activities, antioxidant enzyme activities, H2 O2 levels, and plasma membrane damage. The results indicated an evident relationship between jasmonic acid and the melatonin biosynthesis pathway. Moreover, it seems that the MYC2-ASMT module might contribute to wheat drought tolerance by regulating melatonin contents.

The melatonin system is expressed in the ovine uterus: effect of the day of the oestrous cycle and undernutrition.

CONTEXT: Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus. AIMS: We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2). METHODS: In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements. KEY RESULTS: We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected. CONCLUSIONS: The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition. IMPLICATIONS: The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

Melatonin regulates dihydrotestosterone formation via its membrane receptor in the epididymal epithelial cells of sheep.

Both melatonin and androgen, which affect sperm fertility, are the important factors in epididymis of male animal. In the present study, we confirmed that melatonin regulates the formation of dihydrotestosterone (DHT) in sheep epididymides. Here, we investigated the localization and the expression levels of melatonin keys synthases AANAT and HIOMT, membrane receptors MT1 and MT2, and nuclear receptor RORα in sheep epididymides and testes. We also cultured epididymal epithelial cells and treated them with different concentrations of melatonin (10-11-10-7 M) and luzindole (10-5 M) and 4P-PDOT (10-5 M) to investigate whether melatonin is involved in the regulation of DHT formation and whether these effects are mediated through its receptor pathways. The results showed that AANAT, HIOMT, MT1, MT2, and RORα were differentially expressed between sheep epididymides and testes. In addition, melatonin is involved in mediating the formation of DHT in epididymal epithelial cells, and its influence on DHT is at least partially regulated by the melatonin receptor pathway. Our findings showed that melatonin regulates the functions of the testes and epididymides through an autocrine mechanism and regulates the formation of androgen in sheep epididymides via the receptor pathway. These results provide a basis for further exploring the regulatory mechanisms of melatonin in animal reproduction.

Ectopic expression of HIOMT improves tolerance and nitrogen utilization efficiency in transgenic apple under drought stress.

Melatonin enhances plant tolerance to various environmental stressors. Although exogenous application of melatonin has been investigated, the role of endogenous melatonin metabolism in the response of apples to drought stress and nutrient utilization remains unclear. Here, we investigated the effects of ectopically expressing the human melatonin synthase gene HIOMT on transgenic apple plants under drought stress conditions. The tolerance of transgenic apple lines that ectopically expressed HIOMT improved significantly under drought conditions. After 10 days of natural drought stress treatment, the transgenic apple plants showed higher relative water content, chlorophyll levels and Fv/Fm, and lower relative electrolyte leakage and hydrogen peroxide accumulation, than wild-type plants. The activities of peroxidase, superoxide dismutase and catalase, as well as genes in the ascorbate-glutathione cycle, increased more in transgenic apple plants than in the wild-type. The ectopic expression of HIOMT also markedly alleviated the inhibitory effects of long-term drought stress on plant growth, photosynthetic rate and chlorophyll concentrations in apple plants. The uptake and utilization of 15N increased markedly in the transgenic lines under long-term moderate drought stress. Drought stress sharply reduced the activity of enzymes involved in nitrogen metabolism, but ectopic expression of HIOMT largely reversed that response. The expression levels of genes of nitrogen metabolism and uptake were more upregulated in transgenic apple plants than the wild-type. Overall, our study demonstrates that ectopic expression of HIOMT enhanced the tolerance of apple plants to drought stress, and transgenic apple plants showed improved growth due to higher nutrient utilization efficiency under drought conditions.

MEL-Index: Estimation of Tissue Melatonin Levels Using Gene Expression Data.

Melatonin synthesis by extrapineal sources adjusts physiological and pathophysiological processes in several types of cells and tissues. As measuring locally produced melatonin in fresh tissues might be a challenge due to limited material availability, we created a simple predictive model, the MEL-Index, which infers the content of tissue melatonin using gene expression data. The MEL-Index can be a powerful tool to study the role of melatonin in different contexts. Applying the MEL-Index method to RNA-seq datasets, we have shed light into the clinical relevance of melatonin as a modulator tumor progression and lung infection due to COVID-19. The MEL-Index combines the z-normalized expressions of ASMT (Acetylserotonin O-Methyltransferase), last enzyme of the biosynthetic pathway, and CYP1B1 (cytochrome P450 family enzyme), which encodes the enzyme that metabolizes melatonin in extrahepatic tissues. In this chapter, we describe the steps for calculating the MEL-Index.

Melatonin Synthesis Enzymes Activity: Radiometric Assays for AANAT, ASMT, and TPH.

Melatonin is synthesized and secreted by the pineal gland in mammals. Its synthesis is triggered at night by norepinephrine released in the interstices of the gland. This nocturnal production is dependent on the transcription, translation, and/or activation of the enzymes arylalkylamine-N-acetyltransferase (AANAT), acetylserotonin O-methyltransferase (ASMT), and tryptophan hydroxylase (TPH). In this chapter, the methodology for the analysis of AANAT, ASMT, and TPH activities by radiometric assays will be presented. Several papers were published by our group utilizing these methodologies, evaluating the enzymes modulation by voltage-gated calcium channels, angiotensin II, insulin, anhydroecgonine methyl ester (AEME, crack-cocaine product), ethanol, monosodium glutamate (MSG), signaling pathways such as NFkB, and pathophysiological conditions such as diabetes.

Melatonin through blockade of Hif-1α signaling mediates the anti-fibrosis under hypoxia in canine Sertoli cells.

The hypoxic microenvironment of cryptorchidism is an important factor in the impairment and fibrosis of Sertoli cells which result in blood-testis barrier (BTB) destruction and spermatogenesis loss. Recent studies have shown that melatonin, a well-known pineal hormone exerts beneficial effects against pathological fibrosis in a various of organs. However, it is still unknown whether melatonin can regulate hypoxia-induced fibrosis of Sertoli cells. In this study we evaluate melatonin levels, and its synthesizing enzymes, AANAT and HIOMT expression patterns in canine cryptorchidism and contralateral normal testis. Results show abdominal testes presented low melatonin levels and AANAT and HIOMT expression compared with testes located in the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl2) and found that melatonin inhibited the EMT markers expression and ECM production as well as Hif-1α expression of Sertoli cells in a dose-dependent manner. Furthermore, use of Lificiguat (synonyms YC-1, Hif-1α inhibitor) to interfere with the Hif-1α pathway showed a similar effect with melatonin suppression of the fibrosis in Sertoli cells. The results indicate that melatonin supplementation can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with regulating the inhibition of Hif-1α signaling.

Brain Damage-linked ATP Promotes P2X7 Receptors Mediated Pineal N-acetylserotonin Release.

The pineal gland is a key player in surveillance and defense responses. In healthy conditions, nocturnal circulating melatonin (MEL) impairs the rolling and adhesion of leukocytes to the endothelial layer. Fungi, bacteria, and pro-inflammatory cytokines block nocturnal pineal MEL synthesis, facilitating leukocyte migration to injured areas. ATP is a cotransmitter of the noradrenergic signal and potentiates noradrenaline (NAd)-induced MEL synthesis via P2Y1 receptor (P2Y1R) activation. Otherwise, ATP low-affinity P2X7 receptor (P2X7R) activation impairs N-acetylserotonin (NAS) into MEL conversion in NAd incubated pineals. Here we mimicked a focal increase of ATP by injecting low (0.3 and 1.0 µg) and high (3.0 µg) ATP in the right lateral ventricle of adult rats. Nocturnal pineal activity mimicked the in culture data. Low ATP doses increased MEL output, while high ATP dose and the P2X7R agonist BzATP (15.0-50.0 ng) increased NAS pineal and blood content. In the brain, the response was structure-dependent. There was an increase in cortical and no change in cerebellar MEL. These effects were mediated by changes in the expression of coding genes to synthetic and metabolizing melatonergic enzymes. Thus, the pineal gland plays a role as a first-line structure to respond to the death of cells inside the brain by turning NAS into the darkness hormone.

Effects of SNPs in AANAT and ASMT Genes on Milk and Peripheral Blood Melatonin Concentrations in Holstein Cows (Bos taurus).

Aralkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), the two rate-limiting enzymes for melatonin synthesis, regulate melatonin production in mammals. Through analysis of the milk melatonin level and dairy herd improvement (DHI) index, it was found that the melatonin concentration in milk was significantly negatively correlated with the 305 day milk yield (305M) and peak milk yield (PeakM) (p < 0.05), while it was significantly positively correlated with the serum melatonin concentration (p < 0.05). The full-length of AANAT and ASMT were sequenced and genotyped in 122 cows. Three SNPs in AANAT and four SNPs in ASMT were significantly related to MT levels in the milk and serum (p < 0.05). The SNPs in AANAT were temporarily denoted as N-SNP1 (g.55290169 T>C), N-SNP2 (g.55289357 T>C), and N-SNP3 (g.55289409 C>T). The SNPs in ASMT were temporarily denoted as M-SNP1 (g.158407305 G>A), M-SNP2 (g.158407477 A>G), M-SNP3 (g.158407874 G>A), and M-SNP4 (g.158415342 T>C). The M-SNP1, M-SNP2, and M-SNP3 conformed to the Hardy−Weinberg equilibrium (p > 0.05), while other SNPs deviated from the Hardy−Weinberg equilibrium (p < 0.05). The potential association of MT production and each SNP was statistically analyzed using the method of linkage disequilibrium (LD). The results showed that N-SNP2 and N-SNP3 had some degree of LD (D' = 0.27), but M-SNP1 and M-SNP2 had a strong LD (D' = 0.98). Thus, the DHI index could serve as a prediction of the milk MT level. The SNPs in AANAT and ASMT could be used as potential molecular markers for screening cows to produce high melatonin milk.

Gene expression profile associated with Asmt knockout-induced depression-like behaviors and exercise effects in mouse hypothalamus.

Sleep disorder caused by abnormal circadian rhythm is one of the main symptoms and risk factors of depression. As a known hormone regulating circadian rhythms, melatonin (MT) is also namely N-acetyl-5-methoxytryptamine. N-acetylserotonin methyltransferase (Asmt) is the key rate-limiting enzyme of MT synthesis and has been reportedly associated with depression. Although 50-90% of patients with depression have sleep disorders, there are no effective treatment ways in the clinic. Exercise can regulate circadian rhythm and play an important role in depression treatment. In the present study, we showed that Asmt knockout induced depression-like behaviors, which were ameliorated by swimming exercise. Moreover, swimming exercise increased serum levels of MT and 5-hydroxytryptamine (5-HT) in Asmt knockout mice. In addition, the microarray data identified 10 differentially expressed genes (DEGs) in KO mice compared with WT mice and 29 DEGs in KO mice after swimming exercise. Among the DEGs, the direction and magnitude of change in epidermal growth factor receptor pathway substrate 8-like 1 (Eps8l1) and phospholipase C-ß 2 (Plcb2) were confirmed by qRT-PCR partly. Subsequent bioinformatic analysis showed that these DEGs were enriched significantly in the p53 signaling pathway, long-term depression and estrogen signaling pathway. In the protein-protein interaction (PPI) networks, membrane palmitoylated protein 1 (Mpp1) and p53-induced death domain protein 1 (Pidd1) were hub genes to participate in the pathological mechanisms of depression and exercise intervention. These findings may provide new targets for the treatment of depression.

Melatonin relieves Th17/CD4-CD8- T cells inflammatory responses via nuclear-receptor dependent manner in peripheral blood of primary Sjögren's syndrome.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is a complex systemic autoimmune disease whose clinical spectrum extends from sicca syndrome to systemic involvement. T helper 17 cells (Th17) and CD4-CD8- (double negative, DN) T cells are actively involved in the pathogenesis of pSS. Melatonin shows important immunoregulatory functions in multiple T cell-mediated autoimmune diseases. However, the effects of melatonin on the immune cells of pSS patients are unclear. Hence, this study was aimed to evaluate the effects of melatonin on the immune responses of peripheral pathogenic Th17 and DN T cells from pSS patients, and explore the underlying receptor-related mechanism. METHODS: The concentration of serum and saliva melatonin of pSS patients and healthy controls (HCs) were detected using Enzyme-linked immunosorbent assays (ELISA). Expression of arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT) were conducted in labial glands samples by immunohistochemistry. The mechanism underlying the effects of melatonin on Th17 and DN T cells responses in peripheral blood from pSS was investigated by quantitative real-time polymerase chain reaction (RT-PCR), flow cytometry, ELISA, cell viability, and proliferation assays. RESULTS: Serum and saliva melatonin levels were lower in pSS patients than in HCs, which were negatively correlated with disease activity. The expression levels of melatonin's biosynthetic enzymes (AANAT, HIOMT) and nuclear receptors (RORα, RORγ) were significantly increased in peripheral blood mononuclear cells (PBMCs) from pSS patients. Furthermore, in vitro melatonin administration decreased the expression of melatonin effector/receptor system in peripheral blood of pSS patients. More importantly, Melatonin inhibited pathogenic responses of peripheral Th17 and DN T cells in PBMCs from pSS, which was independent of melatonin membrane receptors. However, melatonin nuclear receptor antagonist SR1001 enhanced the inhibitory ability of melatonin on Th17 and DN T cells production, and agonist SR1078 weakened the effects of melatonin. Additionally, overexpression of the melatonin effector/receptor system in pSS patients appeared to be involved in the disease, due to that melatonin effector/receptor system expression was correlated with the frequency of Th17 or DN T cells. CONCLUSION: Melatonin relieved the inflammatory responses of Th17 and DN T cells in PBMCs from pSS patients in a nuclear receptors-dependent manner,suggesting that melatonin might be beneficial to pSS.

Melatonin: A mitochondrial resident with a diverse skill set.

Melatonin is an ancient molecule that originated in bacteria. When these prokaryotes were phagocytized by early eukaryotes, they eventually developed into mitochondria and chloroplasts. These new organelles retained the melatonin synthetic capacity of their forerunners such that all present-day animal and plant cells may produce melatonin in their mitochondria and chloroplasts. Melatonin concentrations are higher in mitochondria than in other subcellular compartments. Isolated mouse oocyte mitochondria form melatonin when they are incubated with serotonin, a necessary precursor. Oocyte mitochondria subsequently give rise to these organelles in all adult vertebrate cells where they continue to synthesize melatonin. The enzymes that convert serotonin to melatonin, i.e., arylalkylamine-N-acetyltransferase (AANAT) and acetylserotonin-O-methyltransferase, have been identified in brain mitochondria which, when incubated with serotonin, also form melatonin. Melatonin is a potent antioxidant and anti-cancer agent and is optimally positioned in mitochondria to aid in the maintenance of oxidative homeostasis and to reduce cancer cell transformation. Melatonin stimulates the transfer of mitochondria from healthy cells to damaged cells via tunneling nanotubes. Melatonin also regulates the major NAD+-dependent deacetylase, sirtuin 3, in the mitochondria. Disruptions of mitochondrial melatonin synthesis may contribute to a number of mitochondria-related diseases, as discussed in this review.

The Improved Milk Quality and Enhanced Anti-Inflammatory Effect in Acetylserotonin-O-methyltransferase (ASMT) Overexpressed Goats: An Association with the Elevated Endogenous Melatonin Production.

BACKGROUND: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. METHODS: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase (ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. RESULTS: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. CONCLUSIONS: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.

ß-Estradiol inhibits melatonin synthesis and melatonin receptor expression in sheep granulosa cells.

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of ß-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process.

Overexpression of MdASMT9, an N-acetylserotonin methyltransferase gene, increases melatonin biosynthesis and improves water-use efficiency in transgenic apple.

Improving apple water-use efficiency (WUE) is increasingly desirable in the face of global climate change. Melatonin is a pleiotropic molecule that functions in plant development and stress tolerance. In apple, exogenous application of melatonin has been largely investigated, but melatonin biosynthesis and its physiological roles remain elusive. In the plant biosynthetic pathway of melatonin, the last and key step is that N-acetylserotonin methyltransferase (ASMT) converts N-acetylserotonin into melatonin. Here, we identified an apple ASMT gene, MdASMT9, using homology-based cloning and in vitro enzyme assays. Overexpression of MdASMT9 significantly increased melatonin accumulation in transgenic apple lines. Moreover, an enhanced WUE was observed in the MdASMT9-overexpressing apple lines. Under well-watered conditions, this increase in WUE was attributed to an enhancement of photosynthetic rate and stomatal aperture via a reduction in abscisic acid biosynthesis. By contrast, under long-term moderate water deficit conditions, regulations in photoprotective mechanisms, stomatal behavior, osmotic adjustment and antioxidant activity enhanced the WUE in transgenic apple lines. Taken together, our findings shed light on the positive effect of MdASMT9 on improving WUE of apple by modulating melatonin biosynthesis.

Expression of arylalkylamine n-acetyltransferase (AANAT) and acetylserotonin o-methyltransferase (ASMT) in the corpus luteum of pregnant sows and synthesis of melatonin in luteal cells.

In vertebrates, melatonin is mainly synthesized from serotonin in the pineal gland. Many reports have documented that melatonin is also synthesized in the extra-pineal tissues, but the synthesis of melatonin in the corpus luteum (CL) of pregnant sows has never been studied. The objectives of this study were to evaluate the expression of melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), in the CL of sows during pregnancy and to investigate the synthesis of melatonin in luteal cells. Results showed that AANAT and ASMT were both expressed in the CL of sows during pregnancy, higher levels were observed in the early- and mid-stage CL, and the lowest abundance was found in the regressing CL (later-stage). The immunostaining for AANAT and ASMT was predominantly localized in the large luteal cells of porcine CL during pregnancy. Furthermore, melatonin was synthesized in luteal cells from serotonin in a dose- and time-dependent manner. And the expressions of AANAT and ASMT were upregulated by serotonin in luteal cells. In addition, progesterone (P4) secretion and cell viability were promoted in luteal cells treated with serotonin, and the stimulatory effects were blocked by luzindole (a non-selective MT1 and MT2 antagonist). Finally, the expressions of MT1 and MT2 were augmented by serotonin in luteal cells. In conclusion, this study demonstrates for the first time the developmental expression of AANAT and ASMT in the CL and a local synthesis of melatonin in luteal cells of pregnant sows, and suggests a paracrine and/or autocrine role for melatonin in luteal function.

The Reserve/Maximum Capacity of Melatonin's Synthetic Function for the Potential Dimorphism of Melatonin Production and Its Biological Significance in Mammals.

In this article, we attempt to classify a potential dimorphism of melatonin production. Thus, a new concept of "reserve or maximum capacity of melatonin synthetic function" is introduced to explain the subtle dimorphism of melatonin production in mammals. Considering ASMT/ASMTL genes in the pseudoautosomal region of sex chromosomes with high prevalence of mutation in males, as well as the sex bias of the mitochondria in which melatonin is synthesized, we hypothesize the existence of a dimorphism in melatonin production to favor females, which are assumed to possess a higher reserve capacity for melatonin synthesis than males. Under physiological conditions, this subtle dimorphism is masked by the fact that cells or tissues only need baseline melatonin production, which can be accomplished without exploiting the full potential of melatonin's synthetic capacity. This capacity is believed to exceed the already remarkable nocturnal increase as observed within the circadian cycle. However, during aging or under stressful conditions, the reserve capacity of melatonin's synthetic function is required to be activated to produce sufficiently high levels of melatonin for protective purposes. Females seem to possess a higher reserve/maximum capacity for producing more melatonin than males. Thus, this dimorphism of melatonin production becomes manifest and detectable under these conditions. The biological significance of the reserve/maximum capacity of melatonin's synthetic function is to improve the recovery rate of organisms from injury, to increase resistance to pathogen infection, and even to enhance their chances of survival by maximizing melatonin production under stressful conditions. The higher reserve/maximum capacity of melatonin synthesis in females may also contribute to the dimorphism in longevity, favoring females in mammals.