hydroxyindole-O-methyltransferase Research Articles

Ovarian overexpression of ASMT gene increases follicle numbers in transgenic sheep: Association with lipid metabolism

Melatonin plays an important role in mammalian reproductive activities, to further understand the effects of endogenous melatonin on functions of ovary, the transgenic sheep with overexpression of melatonin synthetic enzyme gene ASMT in ovary were generated. The results showed that total melatonin content in follicular fluid of transgenic sheep was significantly greater than that in the wild type. Accordingly, the follicle numbers of transgenic sheep were also significantly greater than those in the WT. The results of follicular fluid metabolites sequencing showed that compared with WT, the differential metabolites of the transgenic sheep were significantly enriched in several signaling pathways, the largest number of metabolites was lipid metabolism pathway and the main differential metabolites were lipids and lipoid molecules. SMART-seq2 were used to analyze the oocytes and granulosa cells of transgenic sheep and WT sheep. The main differential enrichment pathway was metabolic pathway, in which lipid metabolism genes accounted for the majority. In conclusion, this is the first report to show that ovary overexpression of ASMT increased local melatonin production and follicle numbers. These results may imply that ASMT plays an important role in follicle development and formation, and melatonin intervention may be a potential method to promote this process.

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

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 mousemodel, 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 apotential 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.

Systematic Identification and Functional Analysis of the Hypericum perforatum L. bZIP Gene Family Indicating That Overexpressed HpbZIP69 Enhances Drought Resistance.

Basic leucine zipper (bZIP) transcription factors play significant roles in plants' growth and development processes, as well as in response to biological and abiotic stresses. Hypericum perforatum is one of the world's top three best-selling herbal medicines, mainly used to treat depression. However, there has been no systematic identification or functional analysis of the bZIP gene family in H. perforatum. In this study, 79 HpbZIP genes were identified. Based on phylogenetic analysis, the HpbZIP gene family was divided into ten groups, designated A-I and S. The physicochemical properties, gene structures, protein conserved motifs, and Gene Ontology enrichments of all HpbZIPs were systematically analyzed. The expression patterns of all genes in different tissues of H. perforatum (i.e., root, stem, leaf, and flower) were analyzed by qRT-PCR, revealing the different expression patterns of HpbZIP under abiotic stresses. The HpbZIP69 protein is localized in the nucleus. According to the results of the yeast one-hybrid (Y1H) assays, HpbZIP69 can bind to the HpASMT2 (N-acetylserotonin O-methyltransferase) gene promoter (G-box cis-element) to activate its activity. Overexpressing HpbZIP69 in Arabidopsis wild-type lines enhanced their tolerance to drought. The MDA and H2O2 contents were significantly decreased, and the activity of superoxide dismutase (SOD) was considerably increased under the drought stress. These results may aid in additional functional studies of HpbZIP transcription factors, and in cultivating drought-resistant medicinal plants.

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.

Straw Mulch Induced Indoleamines Alleviate Reproductive Depression in Cold Sensitive Hazelnut Cultivars.

Climate change is forcing physiological changes, especially in temperate trees, in which the reproduction phase has been affected harshly, eventually resulting in poor performance. Erratic fluctuations during the flowering periods, predominantly in cold-sensitive, yet industry-desired (sourced), hazelnut cultivars have been causing at least a 10-fold decline in the nut yield. Indoleamines have been noted to provide protection during such abiotic stress conditions. In this study, we investigated the potential involvement of the indoleamine pathway in countering reproductive depression in cold-sensitive hazelnuts by blanketing the ground with wheat straw mulch. The female flower ratio; titers of tryptophan, serotonin, and melatonin; and indoleamine pathway gene regulation were the endpoints for assessing the effects of straw mulch. In the preceding year, we noted that the occurrence of phenological events through the modulation of indoleamines was necessitated via percolation of snowmelt into the rootzone. Otherwise, reproductive depression was noted, especially in harsh conditions, such as 'no snow' or when the rootzone was covered with a plastic sheet to disallow water percolation. When cold-sensitive hazelnut cultivars that were subjected to such deleterious treatments in the preceding years' experiments were treated with straw mulch, the female flower ratio was unaffected and remained on par with that of the cold-hardy locally adapted cultivars. Tryptophan accumulation improved in the (cold-sensitive) sourced cultivars treated with straw mulch and was available as serotonin to counter the cold stress. Lower titers of melatonin explained the slight improvement in female ratio in the sourced cultivars blanketed with straw mulch. ASMT gene regulation via straw mulch treatment emphasized its role in abiotic stress mitigation. A negative trend was noted when improved flowering was compared to the decreased expression of the ASMT gene. Horticultural changes, such as mulch, should provide mitigating solutions to relieve reproductive depression in cold-sensitive hazelnuts, alongside implications in other horticultural crops. The indoleamine toolkit (cellular markers) developed in this study provides insights into the mechanisms of cold sensitivity (abiotic stress) and plausible solutions, such as exogenous application of indoleamines, to propagate climate resilient plant materials with an enhanced capacity to mitigate abiotic stress conditions.

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.

Role of melatonin in enhancing arbuscular mycorrhizal symbiosis and mitigating cold stress in perennial ryegrass (Lolium perenne L.).

Melatonin is a biomolecule that affects plant development and is involved in protecting plants from environmental stress. However, the mechanisms of melatonin's impact on arbuscular mycorrhizal (AM) symbiosis and cold tolerance in plants are still unclear. In this research, AM fungi inoculation and exogenous melatonin (MT) were applied to perennial ryegrass (Lolium perenne L.) seedlings alone or in combination to investigate their effect on cold tolerance. The study was conducted in two parts. The initial trial examined two variables, AM inoculation, and cold stress, to investigate the involvement of the AM fungus Rhizophagus irregularis in endogenous melatonin accumulation and the transcriptional levels of its synthesis genes in the root system of perennial ryegrass under cold stress. The subsequent trial was designed as a three-factor analysis, encompassing AM inoculation, cold stress, and melatonin application, to explore the effects of exogenous melatonin application on plant growth, AM symbiosis, antioxidant activity, and protective molecules in perennial ryegrass subjected to cold stress. The results of the study showed that compared to non-mycorrhizal (NM) plants, cold stress promoted an increase in the accumulation of melatonin in the AM-colonized counterparts. Acetylserotonin methyltransferase (ASMT) catalyzed the final enzymatic reaction in melatonin production. Melatonin accumulation was associated with the level of expression of the genes, LpASMT1 and LpASMT3. Treatment with melatonin can improve the colonization of AM fungi in plants. Simultaneous utilization of AM inoculation and melatonin treatment enhanced the growth, antioxidant activity, and phenylalanine ammonia-lyase (PAL) activity, while simultaneously reducing polyphenol oxidase (PPO) activity and altering osmotic regulation in the roots. These effects are expected to aid in the mitigation of cold stress in Lolium perenne. Overall, melatonin treatment would help Lolium perenne to improve growth by promoting AM symbiosis, improving the accumulation of protective molecules, and triggering in antioxidant activity under cold stress.

Genetic and neural mechanisms of sleep disorders in children with autism spectrum disorder: a review.

The incidence of sleep disorders in children with autism spectrum disorder (ASD) is very high. Sleep disorders can exacerbate the development of ASD and impose a heavy burden on families and society. The pathological mechanism of sleep disorders in autism is complex, but gene mutations and neural abnormalities may be involved. In this review, we examined literature addressing the genetic and neural mechanisms of sleep disorders in children with ASD. The databases PubMed and Scopus were searched for eligible studies published between 2013 and 2023. Prolonged awakenings of children with ASD may be caused by the following processes. Mutations in the MECP2, VGAT and SLC6A1 genes can decrease GABA inhibition on neurons in the locus coeruleus, leading to hyperactivity of noradrenergic neurons and prolonged awakenings in children with ASD. Mutations in the HRH1, HRH2, and HRH3 genes heighten the expression of histamine receptors in the posterior hypothalamus, potentially intensifying histamine's ability to promote arousal. Mutations in the KCNQ3 and PCDH10 genes cause atypical modulation of amygdala impact on orexinergic neurons, potentially causing hyperexcitability of the hypothalamic orexin system. Mutations in the AHI1, ARHGEF10, UBE3A, and SLC6A3 genes affect dopamine synthesis, catabolism, and reuptake processes, which can elevate dopamine concentrations in the midbrain. Secondly, non-rapid eye movement sleep disorder is closely related to the lack of butyric acid, iron deficiency and dysfunction of the thalamic reticular nucleus induced by PTCHD1 gene alterations. Thirdly, mutations in the HTR2A, SLC6A4, MAOA, MAOB, TPH2, VMATs, SHANK3, and CADPS2 genes induce structural and functional abnormalities of the dorsal raphe nucleus (DRN) and amygdala, which may disturb REM sleep. In addition, the decrease in melatonin levels caused by ASMT, MTNR1A, and MTNR1B gene mutations, along with functional abnormalities of basal forebrain cholinergic neurons, may lead to abnormal sleep-wake rhythm transitions. Our review revealed that the functional and structural abnormalities of sleep-wake related neural circuits induced by gene mutations are strongly correlated with sleep disorders in children with ASD. Exploring the neural mechanisms of sleep disorders and the underlying genetic pathology in children with ASD is significant for further studies of therapy.

Genome-wide analysis for the melatonin trait associated genes and SNPs in dairy goat (Capra hircus) as the molecular breeding markers.

Previous studies have reported that the endogenous melatonin level is positively associated with the quality and yield of milk of cows. In the current study, a total of 34,921 SNPs involving 1,177 genes were identified in dairy goats by using the whole genome resequencing bulked segregant analysis (BSA) analysis. These SNPs have been used to match the melatonin levels of the dairy goats. Among them, 3 SNPs has been identified to significantly correlate with melatonin levels. These 3 SNPs include CC genotype 147316, GG genotype 147379 and CC genotype 1389193 which all locate in the exon regions of ASMT and MT2 genes. Dairy goats with these SNPs have approximately 5-fold-higher melatonin levels in milk and serum than the average melatonin level detected in the current goat population. If the melatonin level impacts the milk production in goats as in cows, the results strongly suggest that these 3 SNPs can serve as the molecular markers to select the goats having the improved milk quality and yield. This is a goal of our future study.

Artificial light at night triggers slight transcriptomic effects on melatonin signaling but not synthesis in tadpoles of two anuran species

The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N-acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

Genome Identification of the Tea Plant (Camellia sinensis) ASMT Gene Family and Its Expression Analysis under Abiotic Stress.

The tea plant (Camellia sinensis (L.) O. Ktze) is an important cash crop grown worldwide. It is often subjected to environmental stresses that influence the quality and yield of its leaves. Acetylserotonin-O-methyltransferase (ASMT) is a key enzyme in melatonin biosynthesis, and it plays a critical role in plant stress responses. In this paper, a total of 20 ASMT genes were identified in tea plants and classified into three subfamilies based on a phylogenetic clustering analysis. The genes were unevenly distributed on seven chromosomes; two pairs of genes showed fragment duplication. A gene sequence analysis showed that the structures of the ASMT genes in the tea plants were highly conserved and that the gene structures and motif distributions slightly differed among the different subfamily members. A transcriptome analysis showed that most CsASMT genes did not respond to drought and cold stresses, and a qRT-PCR analysis showed that CsASMT08, CsASMT09, CsASMT10, and CsASMT20 significantly responded to drought and low-temperature stresses; in particular, CsASMT08 and CsASMT10 were highly expressed under low-temperature stress and negatively regulated in response to drought stress. A combined analysis revealed that CsASMT08 and CsASMT10 were highly expressed and that their expressions differed before and after treatment, which indicates that they are potential regulators of abiotic stress resistance in the tea plant. Our results can facilitate further studies on the functional properties of CsASMT genes in melatonin synthesis and abiotic stress in the tea plant.

The pathway of melatonin biosynthesis in common wheat (Triticum aestivum).

Melatonin (Mel) is a multifunctional biomolecule found in both animals and plants. In plants, the biosynthesis of Mel from tryptophan (Trp) has been delineated to comprise of four consecutive reactions. However, while the genes encoding these enzymes in rice are well characterized no systematic evaluation of the overall pathway has, as yet, been published for wheat. In the current study, the relative contents of six Mel-pathway-intermediates including Trp, tryptamine (Trm), serotonin (Ser), 5-methoxy tryptamine (5M-Trm), N-acetyl serotonin (NAS) and Mel, were determined in 24 independent tissues spanning the lifetime of wheat. These studies indicated that Trp was the most abundant amongthe six metabolites, followed by Trm and Ser. Next, the candidate genes expressing key enzymes involved in the Mel pathway were explored by means of metabolite-based genome-wide association study (mGWAS), wherein two TDC genes, a T5H gene and one SNAT gene were identified as being important for the accumulation of Mel pathway metabolites. Moreover, a 463-bp insertion within the T5H gene was discovered that may be responsible for variation in Ser content. Finally, a ASMT gene was found via sequence alignment against its rice homolog. Validations of these candidate genes were performed by in vitro enzymatic reactions using proteins purified following recombinant expression in Escherichia coli, transient gene expression in tobacco, and transgenic approaches in wheat. Our results thus provide the first comprehensive investigation into the Mel pathway metabolites, and a swift candidate gene identification via forward-genetics strategies, in common wheat.

Circ-ERC2 Is Involved in Melatonin Synthesis by Regulating the miR-125a-5p/MAT2A Axis

The circadian rhythm of melatonin secretion in the pineal gland is highly conserved in vertebrates. Melatonin levels are always elevated at night. Acetylserotonin O-methyltransferase (ASMT) is the last enzyme in the regulation of melatonin biosynthesis (N-acetyl-5-hydroxytryptamine-melatonin). S-adenosylmethionine (SAM) is an important methyl donor in mammals and can be used as a substrate for the synthesis of melatonin. Methionine adenosyltransferase (MAT) catalyzes the synthesis of SAM from methionine and ATP and has a circadian rhythm. CircRNA is an emerging type of endogenous noncoding RNA with a closed loop. Whether circRNAs in the pineal gland can participate in the regulation of melatonin synthesis by binding miRNAs to target mat2a as part of the circadian rhythm is still unclear. In this study, we predicted the targeting relationship of differentially expressed circRNAs, miRNAs and mRNAs based on the results of rat pineal RNA sequencing. Mat2a siRNA transfection confirmed that mat2a is involved in the synthesis of melatonin. Circ-ERC2 and miR-125a-5p were screened out by software prediction, dual-luciferase reporter experiments, cell transfection, etc. Finally, we constructed a rat superior cervical ganglionectomy model (SCGx), and the results showed that circ-ERC2 could participate in the synthesis of melatonin through the miR-125a-5p/MAT2A axis. The results of the study revealed that circ-ERC2 can act as a molecular sponge of miR-125a-5p to regulate the synthesis of melatonin in the pineal gland by targeting mat2a. This experiment provides a basis for research on the circadian rhythm of noncoding RNA on pineal melatonin secretion.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned, ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

BackgroundSCNT (somatic cell nuclear transfer) is of great significance to biological research and also to the livestock breeding. However, the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods. This indicates the potential epigenetic variations between them. DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences. In this study, ASMT (acetylserotonin-O-methyltransferase) ovarian overexpression transgenic goat was produced by using SCNT. To investigate whether there are epigenetic differences between cloned and WT (wild type) goats, WGBS (whole-genome bisulfite sequencing) was used to measure the whole-genome methylation of these animals.ResultsIt is observed that the different mCpG sites are mainly present in the intergenic and intronic regions between cloned and WT animals, and their CG-type methylation sites are strongly correlated. DMR (differentially methylated region) lengths are located around 1000 bp, mainly distributed in the exonic, intergenic and intronic functional domains. A total of 56 and 36 DMGs (differentially methylated genes) were identified by GO and KEGG databases, respectively. Functional annotation showed that DMGs were enriched in biological-process, cellular-component, molecular-function and other signaling pathways. A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.ConclusionThe differences in methylation genes among the tested animals have been identified. A total of 10 DMGs associated with growth and development were identified between cloned and WT animals. The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT. These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology, particularly in goats.

Essential oils nano-emulsion confers resistance against Penicillium digitatum in 'Newhall' navel orange by promoting phenylpropanoid metabolism

Inducing natural resistance against pathogen infection in postharvest citrus by exogenous elicitor is a promising alternative to reduce postharvest losses. Here, the potential induced resistance mechanism of blended cinnamaldehyde, carvacrol and eugenol nano-emulsion against P. digitatum in ‘Newhall’ navel orange (Citrus sinensis (L.) Osbeck) was evaluated through combined transcriptomic and metabolomics analysis. Application of nano-emulsion maintained fruit firmness after P. digitatum inoculation, nano-emulsion reduced H2O2 and MDA accumulation of citrus fruit, increased PAL, C4H, 4CL and CAD activity, and higher contents of flavonoids, lignin and total phenolic compounds. Furthermore, higher activities of SOD, CAT, POD and APX in nano-emulsion treated fruit possibly benefited reducing ROS accumulation and lipid peroxidation. The activities of the defense enzymes CHI, GLU were higher in the nano-emulsion treated fruits. RNA-seq identified 653 differentially expressed genes (DEGs) between the control and the nano-emulsion-treated fruit at 48 hpi, including 444 up-regulated and 209 downregulated genes. Genes encoding shikimate O-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid 3-O-methyltransferase / acetylserotonin O-methyltransferase (COMT), cinnamyl-alcohol dehydrogenase (CAD) and peroxidase (POD) were differentially expressed by nano-emulsion treatment. In addition, 175 differential accumulated metabolites between the control and the nano-emulsion-treated fruit at 48 hpi also were identified, the different accumulated metabolites from phenylpropanoid biosynthesis pathway related with disease resistance including L-phenylalanine, caffeic acid, caffeoyl quinic acid, coniferyl alcohol, coniferaldehyde. The integrated transcriptomic and metabolomic profiling indicated that the differentially expressed genes (DEGs) and the differentially accumulated metabolites (DAMs) were mainly involved in phenylpropanoid biosynthesis pathway. Besides, the nano-emulsion significantly induced accumulation of primary metabolites including amino acids, soluble sugars, organic acids, lipids, sugar and alcohols. All these results indicated that blended essential oil nano-emulsion as an antifungal delivery system to induce resistance against P. digitatum infection of ‘Newhall’ navel orange by promoting phenylpropanoid metabolism.

Ni(II) Ions May Target the Entire Melatonin Biosynthesis Pathway—A Plausible Mechanism of Nickel Toxicity

Nickel is toxic to humans. Its compounds are carcinogenic. Furthermore, nickel allergy is a severe health problem that affects approximately 10–20% of humans. The mechanism by which these conditions develop remains unclear, but it may involve the cleavage of specific proteins by nickel ions. Ni(II) ions cleave the peptide bond preceding the Ser/Thr-Xaa-His sequence. Such sequences are present in all four enzymes of the melatonin biosynthesis pathway, i.e., tryptophan 5-hydroxylase 1, aromatic-l-amino-acid decarboxylase, serotonin N-acetyltransferase, and acetylserotonin O-methyltransferase. Moreover, fragments prone to Ni(II) are exposed on surfaces of these proteins. Our results indicate that all four studied fragments undergo cleavage within tens of hours at pH 8.2 and 37 °C, corresponding with the conditions in the mitochondrial matrix. Since melatonin, a potent antioxidant and anti-inflammatory agent, is synthesized within the mitochondria of virtually all human cells, depleting its supply may be detrimental, e.g., by raising the oxidative stress level. Intriguingly, Ni(II) ions have been shown to mimic hypoxia through the stabilization of HIF-1α protein, but melatonin prevents the action of HIF-1α. Considering all this, the enzymes of the melatonin biosynthesis pathway seem to be a toxicological target for Ni(II) ions.

Distinct GmASMTs are involved in regulating transcription factors and signalling cross-talk across embryo development, biotic, and abiotic stress in soybean.

N-Acetylserotonin O-methyltransferase (ASMT) is the final enzyme involved in melatonin biosynthesis. Identifying the expression of ASMT will reveal the regulatory role in the development and stress conditions in soybean. To identify and characterize ASMT in soybean (GmASMT), we employed genome-wide analysis, gene structure, cis-acting elements, gene expression, co-expression network analysis, and enzyme assay. We found seven pairs of segmental and tandem duplication pairs among the 44 identified GmASMTs by genome-wide analysis. Notably, co-expression network analysis reported that distinct GmASMTs are involved in various stress response. For example, GmASMT3, GmASMT44, GmASMT17, and GmASMT7 are involved in embryo development, heat, drought, aphid, and soybean cyst nematode infections, respectively. These distinct networks of GmASMTs were associated with transcription factors (NAC, MYB, WRKY, and ERF), stress signalling, isoflavone and secondary metabolites, calcium, and calmodulin proteins involved in stress regulation. Further, GmASMTs demonstrated auxin-like activities by regulating the genes involved in auxin transporter (WAT1 and NRT1/PTR) and auxin-responsive protein during developmental and biotic stress. The current study identified the key regulatory role of GmASMTs during development and stress. Hence GmASMT could be the primary target in genetic engineering for crop improvement under changing environmental conditions.

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.