hydroxyindole-O-methyltransferase Research Articles

Serotonin and Melatonin Biosynthesis in Plants: Genome-Wide Identification of the Genes and Their Expression Reveal a Conserved Role in Stress and Development.

Serotonin (Ser) and melatonin (Mel) serve as master regulators of plant growth and development by influencing diverse cellular processes. The enzymes namely, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) catalyse the formation of Ser from tryptophan. Subsequently, serotonin N-acetyl transferase (SNAT) and acetyl-serotonin methyltransferase (ASMT) form Mel from Ser. Plant genomes harbour multiple genes for each of these four enzymes, all of which have not been identified. Therefore, to delineate information regarding these four gene families, we carried out a genome-wide analysis of the genes involved in Ser and Mel biosynthesis in Arabidopsis, tomato, rice and sorghum. Phylogenetic analysis unravelled distinct evolutionary relationships among these genes from different plants. Interestingly, no gene family except ASMTs showed monocot- or dicot-specific clustering of respective proteins. Further, we observed tissue-specific, developmental and stress/hormone-mediated variations in the expression of the four gene families. The light/dark cycle also affected their expression in agreement with our quantitative reverse transcriptase-PCR (qRT-PCR) analysis. Importantly, we found that miRNAs (miR6249a and miR-1846e) regulated the expression of Ser and Mel biosynthesis under light and stress by influencing the expression of OsTDC5 and OsASMT18, respectively. Thus, this study may provide opportunities for functional characterization of suitable target genes of the Ser and Mel pathway to decipher their exact roles in plant physiology.

Molecular Mechanisms Underlying the Biosynthesis of Melatonin and Its Isomer in Mulberry.

Mulberry (Morus alba L.) leaves and fruit are traditional Chinese medicinal materials with anti-inflammatory, immune regulatory, antiviral and anti-diabetic properties. Melatonin performs important roles in the regulation of circadian rhythms and immune activities. We detected, identified and quantitatively analyzed the melatonin contents in leaves and mature fruit from different mulberry varieties. Melatonin and three novel isoforms were found in the Morus plants. Therefore, we conducted an expression analysis of melatonin and its isomer biosynthetic genes and in vitro enzymatic synthesis of melatonin and its isomer to clarify their biosynthetic pathway in mulberry leaves. MaASMT4 and MaASMT20, belonging to class II of the ASMT gene family, were expressed selectively in mulberry leaves, and two recombinant proteins that they expressed catalyzed the conversion of N-acetylserotonin to melatonin and one of three isomers in vitro. Unlike the ASMTs of Arabidopsis and rice, members of the three ASMT gene families in mulberry can catalyze the conversion of N-acetylserotonin to melatonin. This study provides new insights into the molecular mechanisms underlying melatonin and its isomers biosynthesis and expands our knowledge of melatonin isomer biosynthesis.

The role of watermelon caffeic acid O-methyltransferase (ClCOMT1) in melatonin biosynthesis and abiotic stress tolerance

Melatonin is a pleiotropic signaling molecule that regulates plant growth and responses to various abiotic stresses. The last step of melatonin synthesis in plants can be catalyzed by caffeic acid O-methyltransferase (COMT), a multifunctional enzyme reported to have N-acetylserotonin O-methyltransferase (ASMT) activity; however, the ASMT activity of COMT has not yet been characterized in nonmodel plants such as watermelon (Citrullus lanatus). Here, a total of 16 putative O-methyltransferase (ClOMT) genes were identified in watermelon. Among them, ClOMT03 (Cla97C07G144540) was considered a potential COMT gene (renamed ClCOMT1) based on its high identities (60.00–74.93%) to known COMT genes involved in melatonin biosynthesis, expression in almost all tissues, and upregulation under abiotic stresses. The ClCOMT1 protein was localized in the cytoplasm. Overexpression of ClCOMT1 significantly increased melatonin contents, while ClCOMT1 knockout using the CRISPR/Cas-9 system decreased melatonin contents in watermelon calli. These results suggest that ClCOMT1 plays an essential role in melatonin biosynthesis in watermelon. In addition, ClCOMT1 expression in watermelon was upregulated by cold, drought, and salt stress, accompanied by increases in melatonin contents. Overexpression of ClCOMT1 enhanced transgenic Arabidopsis tolerance against such abiotic stresses, indicating that ClCOMT1 is a positive regulator of plant tolerance to abiotic stresses.

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92±70.6074 vs 263.10±34.60 pg/mL, P<.05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

Nitric Oxide Functions as a Downstream Signal for Melatonin-Induced Cold Tolerance in Cucumber Seedlings.

Melatonin (MT) and nitric oxide (NO) are two multifunctional signaling molecules that are involved in the response of plants to abiotic stresses. However, how MT and NO synergize in response to cold stress affecting plants is still not clear. In this study, we found that endogenous MT accumulation under cold stress was positively correlated with cold tolerance in different varieties of cucumber seedlings. The data presented here also provide evidence that endogenous NO is involved in the response to cold stress. About 100 μM MT significantly increased the nitrate reductase (NR) activity, NR-relative messenger RNA (mRNA) expression, and endogenous NO accumulation in cucumber seedlings. However, 75 μM sodium nitroprusside (SNP, a NO donor) showed no significant effect on the relative mRNA expression of tryptophan decarboxylase (TDC), tryptamine-5-hydroxylase (T5H), serotonin-N-acetyltransferase (SNAT), or acetylserotonin O-methyltransferase (ASMT), the key genes for MT synthesis and endogenous MT levels. Compared with H2O treatment, both MT and SNP decreased electrolyte leakage (EL), malondialdehyde (MDA), and reactive oxygen species (ROS) accumulation by activating the antioxidant system and consequently mitigated cold damage in cucumber seedlings. MT and SNP also enhanced photosynthetic carbon assimilation, which was mainly attributed to an increase in the activity and mRNA expression of the key enzymes in the Calvin–Benson cycle. Simultaneously, MT- and SNP-induced photoprotection for both photosystem II (PSII) and photosystem I (PSI) in cucumber seedlings, by stimulating the PsbA (D1) protein repair pathway and ferredoxin-mediated NADP+ photoreduction, respectively. Moreover, exogenous MT and SNP markedly upregulated the expression of chilling response genes, such as inducer of CBF expression (ICE1), C-repeat-binding factor (CBF1), and cold-responsive (COR47). MT-induced cold tolerance was suppressed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a specific scavenger of NO). However, p-chlorophenylalanine (p-CPA, a MT synthesis inhibitor) did not affect NO-induced cold tolerance. Thus, novel results suggest that NO acts as a downstream signal in the MT-induced plant tolerance to cold stress.

Roles of Endogenous Melatonin in Resistance to Botrytis cinerea Infection in an Arabidopsis Model.

Melatonin is an important bioactive molecule in plants. Two synthetases, N-acetylserotonin methyltransferase (ASMT) and serotonin N-acetyltransferase (SNAT) are involved in the final two steps of melatonin synthesis. Melatonin participates in responses to a variety of biotic and abiotic stresses in plants, but few studies have addressed the roles of endogenous melatonin in pathogen resistance. We investigated the role of endogenous melatonin in resistance to Botrytis cinerea infection in an Arabidopsis thaliana model system. Plant lines that overexpressed ASMT or SNAT through genetic manipulation showed upregulated expression of resistance genes PR1 and PR5, transcription factor gene WRKY33, and jasmonic acid (JA) defense pathway marker gene PDF1.2, and downregulated transcription factor gene MYC2 in JA signaling pathway. Higher melatonin content also enhanced the activity of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD), increased JA content, reduced plant disease symptoms, and reduced lesion size in leaves. These findings indicate that endogenous melatonin enhances plant resistance to B. cinerea infection. In contrast, ASMT and SNAT gene silencing lines showed opposite results and were more susceptible to B. cinerea. Thus, it can be demonstrated that melatonin functions as an effective regulator of plant stress resistance at the genetic level. A schematic model is presented for its role in resistance to B. cinerea infection. Our findings also helped to elucidate the associated signal transduction pathways and interactions between melatonin and other plant hormones.

Identification of Serotonin as a Predictive Marker for Breast Cancer Patients.

PurposeCumulative evidence has demonstrated that breast cancer was the most commonly diagnosed cancer in women. Despite growing evidence for a link between serotonin and tumorigenesis, research on the expression of serotoninergic systems in the human breast cancer cell and tissue has only rarely been reported.MethodsFirst, immunofluorescence staining, ELISA and Western blotting were used to detect serotonin and melatoninergic systems in various breast cancer cell types. Then, serotonin expression was evaluated in the cultures of TPBC cell line BT-474 and TNBC cell line MDA-MB-231 using immunofluorescence assay. To further explore the diagnostic role of serotonin in breast cancer, serotonin expression was conducted in the TPBC and TNBC tumor sections by immunostaining analysis.ResultsOur results suggested that both human breast cancer cells and human breast epithelial cell line could synthesize serotonin and melatonin. Unlike melatonin, serotonin levels varied significantly between human breast cancer and breast epithelial cell line (p<0.01). In addition, serotonin N-acetyltransferase (NAT) and acetylserotonin methyltransferase (ASMT), the key enzymes in the pathway of melatonin synthesis from serotonin, were also detectable. In agreement with these findings of human breast cancer cell and human breast epithelial cell line, serotonin expression was also much higher in triple-negative (PR−, ER−, HER-2−) breast cancer (TNBC) and triple-positive breast cancer (TPBC) compared to para-carcinoma tissues (PCTs).ConclusionHere, we provided evidence that the human breast cancer cell (MCF-7, Bcap-37) and human breast epithelial cell (MCF-10A) could synthesize intrinsic serotonin and melatonin, and serotonin expression was higher in the breast cancer tissue compared with PCT. The findings suggested that serotonin might be used as a predictive marker for breast cancer patients.

Melatonergic index as a prognostic biomarker of reproductive organ cancers: correlations with metabolic parameters as well as clock genes PER1 and TIMELESS

Cancers of the reproductive organs are often hard to be detected, and patients’ survival rate drops considerably even when the tumor is removed. Based on the fact that melatonin levels are significantly lower in cancer cells than that in the healthy cells, and this melatonin suppression remains during tumor progression, we have examined a simple two-gene-based melatonergic system [the indices of melatonin synthesis and metabolism (ASMT:CYP1A1, ASMT:CYP1A2, ASMT:CYP1B1)] as a prognostic factor for reproductive organ cancer survival rate. RNA-seq data from The Cancer Genome Atlas (TCGA) of seven types of human reproductive organ tumors (n = 3571 samples) were analyzed. By stratifying the set of index values into high vs low risk, we observed that patients with a high melatonergic index had improved survival rates for cervical, ovarian, and endometrial cancers. Patients at high-risk (low melatonergic index) showed a trend of diagnosis of breast, prostate, and testicular cancers at the younger age, while patients with cervical, ovarian, and endometrial cancers presented with higher tumor staging. The melatonergic indices, especially the ASMT:CYP1B1, positively correlated with the clock gene PER1 while negatively correlated with the clock gene TIMELESS in all reproductive organ cancers. We further analyzed the correlation between the expression profiles of the melatonin-synthesizing enzyme (ASMT gene) with metabolic enzyme-encoding genes. Notably, LDHA, PDK1, and PDHA1 showed a higher correlation in male and female reproductive organ tumors, while IDH1, SDHB, GLS, and ATP1A1 had a positive correlation in breast, testicular, and endometrial cancers. These results have provided a comprehensive evaluation of the melatonergic system in relation to the reproductive organ tumor microenvironment and identified promising gene signatures as potential biomarkers for cancer diagnostics, prognostics, and therapeutics.

Cytoguardin: A Tryptophan Metabolite against Cancer Growth and Metastasis.

Cytoguardin was identified in the conditioned medium of fibroblasts as a tryptophan metabolite, 5-methoxytryptophan (5-MTP). It is synthesized via two enzymatic steps: tryptophan hydroxylase (TPH) and hydroxyindole O-methyltransferase (HIOMT). A truncated HIOMT isoform, HIOMT298, catalyzes 5-MTP synthesis. Cancer cells produce scarce 5-MTP due to defective HIOMT298 expression. 5-MTP inhibits cancer cell COX-2 expression and thereby reduces COX-2-mediated cell proliferation and migration. 5-MTP also inhibits MMP-9 expression and thereby reduces cancer cell invasion. 5-MTP exerts its anti-cancer effect by blocking p38 MAPK and p38-mediated NF-κB and p300 HAT activation. The stable transfection of A549 cells with HIOMT298 restores 5-MTP production which renders cancer cells less aggressive. The implantation of HIOMT-transfected A549 into subcutaneous tissues of a murine xenograft tumor model shows that HIOMT-transduced A549 cells form smaller tumors and generate fewer metastatic lung nodules than control A549 cells. HIOMT298 transfection suppresses aromatic amino acid decarboxylase (AADC) expression and serotonin production. Serotonin is a cancer-promoting factor. By restoring 5-MTP and suppressing serotonin production, HIOMT298 overexpression converts cancer cells into less malignant phenotypes. The analysis of HIOMT expression in a human cancer tissue array showed reduced HIOMT levels in a majority of colorectal, pancreatic, and breast cancer. HIOMT298 may be a biomarker of human cancer progression. Furthermore, 5-MTP has the potential to be a lead compound in the development of new therapy for the chemoprevention of certain cancers such as hepatocellular cancer.

The Synergistic role of serotonin and melatonin during temperature stress in promoting cell division, ethylene and isoflavones biosynthesis in Glycine max

Serotonin (SER) and melatonin (MEL) are signalling molecules involved in signalling in various stress response mechanisms in plants. However, their role and molecular mechanism in the accumulation of isoflavones and biomass under temperature stress are not clearly defined. To explicate their function, SER and MEL at different ratios were applied to the in vitro cultures of soybean at 16, 24, and 32 °C and were analyzed for the accumulation of bioactive compounds, isoflavones content, and culture biomass followed by transcript levels of cell division, isoflavones, and ethylene biosynthesis genes. An increase in biomass, total phenolics, total flavonoids, and different forms of isoflavones content was evident in the treatments. The expression level of critical genes of isoflavone (chalcone synthase, chalcone reductase, chalcone isomerase, and isoflavone synthase), ethylene (s-adenosyl methionine and 1-aminocyclopropane-1-carboxylic acid oxidase), cell division (cyclin and cyclin-dependent kinase), and MEL biosynthesis genes (acetyl serotonin O-methyltransferase and serotonin N-acetyltransferase) and transcription factors (melatonin 2 hydroxylase, myeloblastosis like protein) were co-expressed during temperature stress in the presence of SER and MEL. At 32 °C, the SER and MEL application showed maximum biomass and isoflavones content and was substantiated with the corresponding gene expression. We hypothesize that the SER and MEL ratio may play a role in increasing the biomass and isoflavones content under temperature stress in soybean cell culture.

Diaphorina citri Genome Possesses a Complete Melatonin Biosynthesis Pathway Differentially Expressed under the Influence of the Phytopathogenic Bacterium, Candidatus Liberibacter asiaticus.

Simple SummaryThe indole-like compound, melatonin, is a tryptophan-derivative that is secreted by the pineal gland. Melatonin is ubiquitously distributed in both prokaryotes and eukaryotes including animals and plants. In animals, melatonin plays pleiotropic regulatory roles in several biological and physiological functions including sleep, circadian rhythm, oxidative stress, immune response, aging, apoptosis, and autophagy. Moreover, it might have anti-inflammatory, anti-tumor, and anti-cancer activities. Although most, if not all, of these genes were cloned and characterized previously in numerous animal species, none of them have been identified from the Asian citrus psyllid, Diaphorina citri, in the vector of Huanglongbing yet. In the current study, we performed a genome-wide analysis and introduces a shortlist of six putative melatonin biosynthesis-related genes included two putative tryptophan 5-hydroxylase (DcT5H-1 and DcT5H-2), a putative aromatic amino acid decarboxylase (DcAADC), two putative arylalkylamine N-acetyltransferase (DcAANAT-1 and DcAANAT-2), and putative N-acetylserotonin O-methyltransferase (DcASMT), which could indicate sites of functional or structural constraint. All these genes were differentially expressed under the influence of the phytopathogenic bacterium, Candidatus Liberibacter asiaticus, and after melatonin supplementation. Our findings could be a further step for optimization and cloning of melatonin biosynthesis genes of Diaphorina citri.Melatonin is synthesized from the amino acid L-tryptophan via the shikimic acid pathway and ubiquitously distributed in both prokaryotes and eukaryotes. Although most of melatonin biosynthesis genes were characterized in several plants and animal species including the insect model, Drosophila melanogaster, none of these enzymes have been identified from the Asian citrus psyllid, Diaphorina citri. We used comprehensive in silico analysis and gene expression techniques to identify the melatonin biosynthesis-related genes of D. citri and to evaluate the expression patterns of these genes within the adults of D. citri with gradient infection rates (0, 28, 34, 50, 58, and 70%) of the phytopathogenic bacterium Candidatus Liberibacter asiaticus and after the treatment with exogenous melatonin. We showed that the D. citri genome possesses six putative melatonin biosynthesis-related genes including two putative tryptophan 5-hydroxylase (DcT5H-1 and DcT5H-2), a putative aromatic amino acid decarboxylase (DcAADC), two putative arylalkylamine N-acetyltransferase (DcAANAT-1 and DcAANAT-2), and putative N-acetylserotonin O-methyltransferase (DcASMT). The infection with Ca. L. asiaticus decreased the transcript levels of all predicted genes in the adults of D. citri. Moreover, melatonin supplementation induced their expression levels in both healthy and Ca. L. asiaticus-infected psyllids. These findings confirm the association of these genes with the melatonin biosynthesis pathway.

Des gènes circadiens à la sémiologie psychiatrique

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

A novel signature predicts recurrence risk and therapeutic response in breast cancer patients.

Acetylserotonin O-methyltransferase (ASMT) is a key enzyme in the synthesis of melatonin. Although melatonin has been shown to exhibit anticancer activity and prevents endocrine resistance in breast cancer, the role of ASMT in breast cancer progression remains unclear. In this retrospective study, we analyzed gene expression profiles in 27 data sets on 7244 patients from 11 countries. We found that ASMT expression was significantly reduced in breast cancer tumors relative to healthy tissue. Among breast cancer patients, those with higher levels of ASMT expression had better relapse-free survival outcomes and longer metastasis-free survival times. Following treatment with tamoxifen, patients with greater ASMT expression experienced longer periods before relapse or distance recurrence. Motivated by these results, we devised an ASMT gene signature that can correctly identify low-risk cases with a sensitivity and specificity of 0.997 and 0.916, respectively. This signature was robustly validated using 23 independent breast cancer mRNA array data sets from different platforms (consisting of 5800 patients) and an RNAseq data set from TCGA (comprising 1096 patients). Intriguingly, patients who are classified as high-risk by the signature benefit from adjuvant chemotherapy, and those with grade II tumors who are classified as low-risk exhibit improved overall survival and distance relapse-free outcomes following endocrine therapy. Together, our findings more clearly elucidate the roles of ASMT, provide strategies for improving the efficacy of tamoxifen treatment and help to identify those patients who may maximally benefit from adjuvant or endocrine therapies.

A de novo Transcriptome Assembly of the European Flounder (Platichthys flesus): The Preselection of Transcripts Encoding Active Forms of Enzymes

The RNA sequencing data sets available for different fish species show a potentially high variety of forms of enzymes just in teleosts. This is primarily considered an effect of the first round of whole-genome duplication with mutations in duplicated genes (isozymes) and alternative splicing of mRNA (isoforms). However, the abundance of the mRNA transcript variants is not necessarily reflected in the abundance of active forms of proteins. We have investigated the transcriptional profiles of two enzymes, aralkylamine N-acetyltransferase (AANAT: EC 2.3.1.87) and N-acetylserotonin O-methyltransferase (ASMT: EC 2.1.1.4), in the eyeball, brain, intestines, spleen, heart, liver, head kidney, gonads, and skin of the European flounder (Platichthys flesus). High-throughput next-generation sequencing technology NovaSeq6000 was used to generate 500M sequencing reads. These were then assembled and filtered producing 75k reliable contigs. Gene ontology (GO) terms were assigned to the majority of annotated contigs/unigenes based on the results of PFAM, PANTHER, UniProt, and InterPro protein database searches. BUSCOs statistics for metazoa, vertebrata, and actinopterygii databases showed that the reported transcriptome represents a high level of completeness. In this article, we show how to preselect transcripts encoding the active enzymes (isozymes or isoforms), using AANAT and ASMT in the European flounder as the examples. The data can be used as a tool to design the experiments as well as a basis for discussion of diversity of enzyme forms and their physiological relevance in teleosts.

Melatonin confers heavy metal-induced tolerance by alleviating oxidative stress and reducing the heavy metal accumulation in Exophiala pisciphila, a dark septate endophyte (DSE)

BackgroundMelatonin (MT), ubiquitous in almost all organisms, functions as a free radical scavenger. Despite several reports on its role as an antioxidant in animals, plants, and some microorganisms, extensive studies in filamentous fungi are limited. Based upon the role of melatonin as an antioxidant, we investigated its role in heavy metal-induced stress tolerance in Exophiala pisciphila, a dark septate endophyte (DSE), by studying the underlying mechanisms in alleviating oxidative stress and reducing heavy metal accumulation.ResultsA significant decrease in malondialdehyde (MDA) and oxygen free radical (OFR) in E. pisciphila was recorded under Cd, Zn, and Pb stresses as compared to the control. Pretreatment of E. pisciphila with 200.0 μM exogenous melatonin significantly increased the activity of superoxide dismutase (SOD) under Zn and Pb stresses. Pretreatment with 200.0 μM melatonin also lowered Cd, Zn, and Pb concentrations significantly. Melatonin production was enhanced by Cd, Cu, and Zn after 2 d, and melatonin biosynthetic enzyme genes, E. pisciphila tryptophan decarboxylase (EpTDC1) and serotonin N-acetyltransferase (EpSNAT1), were transcriptionally upregulated. The overexpression of EpTDC1 and N-acetylserotonin O-methyltransferase (EpASMT1) in Escherichia coli and Arabidopsis thaliana enhanced its heavy metal-induced stress tolerance. The overexpression of EpTDC1 and EpASMT1 reduced the Cd accumulation in the whole A. thaliana plants, especially in the roots.ConclusionsMelatonin conferred heavy metal-induced stress tolerance by alleviating oxidative stress, activating antioxidant enzyme SOD, and reducing heavy metal accumulation in E. pisciphila. Melatonin biosynthetic enzyme genes of E. pisciphila also played key roles in limiting excessive heavy metal accumulation in A. thaliana. These findings can be extended to understand the role of melatonin in other DSEs associated with economically important plants and help develop new strategies in sustainable agriculture practice where plants can grow in soils contaminated with heavy metals.

Heat shock protein 90 co-chaperone modules fine-tune the antagonistic interaction between salicylic acid and auxin biosynthesis in cassava

Heat shock protein 90 (HSP90) is an important molecular chaperone in plants. However, HSP90-mediated plant immune response remains elusive in cassava. In this study, cassava bacterial blight (CBB) induces the expression of MeHsf8, which directly targets MeHSP90.9 to activate its expression and immune response. Further identification of SHI-related sequence 1 (MeSRS1) and MeWRKY20 as MeHSP90.9 co-chaperones revealed the underlying mechanism of MeHSP90.9-mediated immune response. MeHSP90.9 interacts with MeSRS1 and MeWRKY20 to promote their transcriptional activation of salicylic acid (SA) biosynthetic gene avrPphB Susceptible 3 (MePBS3) and tryptophan metabolic gene N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively, so as to activate SA biosynthesis but inhibit tryptophan-derived auxin biosynthesis. Notably, genetic experiments confirmed that overexpressing MePBS3 and MeASMT2 could rescue the effects of silencing MeHsf8-MeHSP90.9 on disease resistance. This study highlights the dual regulation of SA and auxin biosynthesis by MeHSP90.9, providing the mechanistic understanding of MeHSP90.9 client partners in plant immunity.

Exogenous phytosulfokine α application delays senescence and promotes antioxidant nutrient accumulation in strawberry fruit during cold storage by triggering endogenous phytosulfokine α signaling

In this study, the mechanism by which the exogenous application of 150 nM signaling bioactive peptide phytosulfokine α (PSKα) delays senescence and improves antioxidant nutrient accumulation in strawberry fruit during storage at 4 °C for 18 d was investigated. Results showed that the higher endogenous accumulation of PSKα in strawberry fruit treated with 150 nM PSKα may result from the higher expression of PSK3 and PSK6 genes. Besides, the higher endogenous accumulation of Ca2+ in strawberry fruit treated with 150 nM PSKα may be ascribed to the higher cytosolic accumulation of cGMP, resulting from the triggering of endogenous PSKα signaling pathway, represented by higher expression of PSKR1 gene. Besides, the higher endogenous melatonin accumulation resulting from higher expression of TDC, T5H, SNAT, and ASMT genes in strawberry fruit treated with 150 nM PSKα may be ascribed to the higher endogenous accumulation of Ca2+. Moreover, the higher ABTS and DPPH scavenging capacity in strawberry fruit treated with 150 nM PSKα may be ascribed to the higher accumulation of phenols, flavonoids, and anthocyanins, resulting from higher gene expression and activities of PAL and CHS. Based on our findings, the exogenous application of PSKα could be employed as a beneficial procedure for delaying senescence and improving antioxidant nutrient accumulation in strawberry fruit during cold storage, by triggering endogenous PSKα signaling, promoting endogenous melatonin accumulation, and activating the phenylpropanoid pathway.

The effects of melatonin treatment on cap browning and biochemical attributes of Agaricus bisporus during low temperature storage

Exogenous melatonin application at 0, 1, 10, 100, and 1000 µM retarded cap browning of button mushrooms (Agaricus bisporus) by 78.35, 31.40, 30.91, 27.17, and 32.50 %, respectively.Mushrooms treated with 100 µM melatonin also had lower weight loss and higher firmness. During the first 5 days of storage at 4 °C, higher H2O2 accumulation may serve as a signal for promoting endogenous melatonin accumulation by triggering the expression of TDC, T5H, SNAT, and ASMT genes, beneficial for preserving membrane integrity. Besides, the higher accumulation of phenols in mushrooms treated with 100 µM melatonin may be ascribed to higher PAL and lower PPO gene expression and enzyme activity. Moreover, higher DPPH scavenging capacity in mushrooms treated with 100 µM melatonin may be ascribed to the higher accumulation of phenols and ascorbic acid.

Key Genes in the Melatonin Biosynthesis Pathway with Circadian Rhythm Are Associated with Various Abiotic Stresses.

Melatonin (N-acetyl-5-methoxytryptamine), a well-known animal hormone, is involved in several biological processes including circadian rhythm and the regulation of abiotic stress. A systematic understanding of the circadian regulation of melatonin biosynthesis-related genes has not been achieved in rice. In this study, key genes for all of the enzymes in the melatonin biosynthetic pathway that showed a peak of expression at night were identified by microarray data analysis and confirmed by qRT–PCR analysis. We further examined the expression patterns of the four genes under drought, salt, and cold stresses. The results showed that abiotic stresses, such as drought, salt, and cold, affected the expression patterns of melatonin biosynthetic genes. In addition, the circadian expression patterns of tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), and serotonin N-acetyltransferase (SNAT) genes in wild-type (WT) plants was damaged by the drought treatment under light and dark conditions. Conversely, N-acetylserotonin O-methyltransferase (ASMT) retained the circadian rhythm. The expression of ASMT was down-regulated by the rice gigantea (OsGI) mutation, suggesting the involvement of the melatonin biosynthetic pathway in the OsGI-mediated circadian regulation pathway. Taken together, our results provide clues to explain the relationship between circadian rhythms and abiotic stresses in the process of melatonin biosynthesis in rice.

Pineal Gland Rhythm and Circadian Physiology

An unpaired neuroendocrine and highly vascularized organ, the pineal gland is located in the middle of the brain nearly in all vertebrates. Its main function is to synthesize the melatonin hormone (N-acetyl-5-methoxytryptamine) in the pinealocytes, which converts light and dark information to complete body physiology. Light inhibits melatonin production whereas darkness stimulates it. Melatonin is synthesized from amino acid tryptophan. Dietary amino acid tryptophan is converted into melatonin in four consecutive biochemical steps.This conversion process includes: conversion of amino acid tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase 1 (TPH1); synthesis of 5-hydroxytryptamine (5-HT or serotonin) by aromatic amino acid decarboxylase; formation of N-acetylserotonin (NAS) by arylalkylamine N-acetyltransferase (AANAT), and production of melatonin by hydroxyindole-O-methyltransferase (HIOMT) (also termed N-acetylserotonin methyltransferase or ASMT).The pineal gland in rat exhibits circadian rhythms of release for all four tryptophan enzymatic derivatives, with larger levels at night. 5-HTP and serotonin are both released at quite high levels during the day and increase much more at night, in contrast to NAS and melatonin, which are hardly detectable during the day